Fluorescent imaging agents

ABSTRACT

Provided is a family of intramolecularly quenched imaging agents for use in both in vivo and in vitro imaging that contain at least one enzymatically cleavable oligopeptide and two fluorophores or a fluorophore and a quencher. When subjected to proteolytic cleavage, at least one fluorophore is unquenched and becomes capable of producing a fluorescent signal upon excitation with light of an appropriate wavelength. Also provided are in vivo and in vitro imaging methods using such imaging agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 61/022,024, filedJan. 18, 2008, the entire contents of which are herein incorporated byreference.

BACKGROUND

Current approaches for assessing of molecular endpoints in diseaseusually require tissue and blood sampling, surgery, and in the case ofexperimental animals, sacrifice at different time points. Despiteimprovements in noninvasive imaging, more sensitive and specific imagingagents and methods are urgently needed. Imaging techniques capable ofvisualizing specific molecular targets and/or entire pathways wouldsignificantly enhance the ability to diagnose and assess treatmentefficacy of therapeutic interventions for many different disease states.Most current imaging techniques report primarily on anatomical orphysiological information (e.g., magnetic resonance imaging (MRI),computed tomography (CT), and ultrasound). Newer modalities, such as,optical imaging and new molecular imaging probes have the potential torevolutionize the way disease is detected, treated, and monitored.

Molecular imaging is a developing field in the imaging sciences thattranscends the traditional boundaries of imaging structure orphysiology, and has the potential to revolutionize current research andclinical practices towards real molecular medicine. The one paradigm formolecular imaging involves the use of a “molecular” probe or agent thatselectively targets a particular gene, protein, receptor or a cellularfunction, with the absence, presence, amount or concentration of thespecific target being indicative of a particular disease state.

In particular, optical imaging offers several strong performanceattributes that make it a truly powerful molecular imaging approach,both in the research and clinical settings. Specifically, opticalimaging is fast, safe, cost effective and highly sensitive. Scan timestypically are on the order of seconds to minutes, there is no ionizingradiation, and the imaging systems are relatively simple to use. Inaddition, optical probes can be designed as dynamic molecular imagingagents that can alter their reporting profiles in vivo to providemolecular and functional information in real time. In order to achievemaximum penetration and sensitivity in vivo, the choice for most opticalimaging in biological systems is within the red and near-infrared (NIR)spectral region, although other wavelengths in the visible region canalso be used. In the NIR wavelength range, absorption by physiologicallyabundant absorbers such as hemoglobin or water is minimized.

Although different types of optical imaging probes have been developedincluding (1) probes that become activated after target contact (e.g.,binding or interaction), (2) wavelength shifting beacons, (3) multicolorfluorescence probes, (4) probes that have high binding affinity totargets, i.e., that remain within a target region while non-specificprobes are cleared from the body (Achilefu et al., Invest. Radiol.,35:479-485, 2000; Becker et al., Nature Biotech. 19:327-331, 2001; Bujaiet al., J. Biomed. Opt. 6:122-133, 2001; Ballou et al. Biotechnol. Prog.13:649-658, 1997; and Neri et al., Nature Biotech. 15:1271-1275, 1997),and (5) fluorescent semiconductor based probes, there is still anongoing need for imaging probes that, for example, are capable ofproviding high quality images and molecular information.

SUMMARY OF THE INVENTION

The present invention provides fluorescent imaging agents that havesignificantly enhanced fluorescent properties upon activation and can beused for in vivo and in vitro imaging applications. The imaging agentshave a fluorescence reporter system, which in certain embodiments,contains two fluorophores or a fluorophore and a quencher, where onefluorophore quenches the other fluorophore or the quencher quenches thefluorophore. Although the imaging agents may have no more than twofluorophores they still have an extinction coefficient sufficient for invivo imaging applications. Furthermore, their size permits the agents tobecome quickly distributed through the tissues or body fluid of asubject, which allows the agent to travel to tissues or cells foractivation by proteases, binding to cell surface receptors orinternalization within cells.

The imaging agents have enhanced fluorescent properties upon activationand under certain circumstances can have magnetic properties, forexample, paramagnetic or superparamagnetic properties, so that theimaging agents can be used as MRI or multi-modality imaging agents (forexample, optical imaging and magnetic resonance imaging). In addition,the imaging agents optionally can include diagnostic and or therapeuticmoieties, for example, radioactive metals, so that the resulting imagingagents can be used like radiopharmaceuticals, nuclear imaging agents ormulti-modality imaging agents (for example, in an optical imaging andnuclear imaging environment).

In one aspect, the invention provides an intramolecularly-quenchedimaging agent. The agent comprises (a) an enzymatically cleavableoligopeptide comprising from about 2 to about 30 amino acid residues;(b) an optional biological modifier chemically linked to theenzymatically cleavable oligopeptide; and (c) either two fluorophores orone fluorophore and one quencher, each covalently linked, directly orindirectly, to the oligopeptide or to the optional biological modifier,wherein one fluorophore quenches the other fluorophore or the quencherquenches the fluorophore and upon enzymatic cleavage of theoligopeptide, at least one fluorophore becomes unquenched and is capableof producing a greater fluorescent signal when excited byelectromagnetic radiation than before enzymatic cleavage of theoligopeptide.

The oligopeptide can comprise from about 2 to about 25 amino acidresidues, from about 2 to about 14 amino acid residues, from about 4 toabout 10 amino acid residues, or from about 5 to about 8 amino acidresidues.

In certain embodiments, the imaging agent can be represented by FormulaI:

wherein:

-   -   B is [M_(m)-ECO-M_(m)];    -   ECO is an enzymatically cleavable oligopeptide;    -   G is L-F;    -   F is a fluorophore or quencher;    -   M is a biological modifier;    -   K is N-L;    -   N is a non-fluorescent reporter;    -   L, independently, for each occurrence, is a linker moiety or a        bond;    -   n is an integer from 0 to 3;    -   m, independently, for each occurrence, is 0 or 1, and optionally        at least one m is 1; and    -   f, independently, for each occurrence, is an integer from 0 to        2, wherein the total number of fluorophores F in the agent is no        greater than 2.

Each F can be chemically linked, directly or through the linker moietyL, to a separate amino acid of the oligopeptide. Alternatively, at leastone F is chemically linked, directly or through the linker moiety L, toM. In certain embodiments, ECO is a cyclic oligopeptide.

In addition, in certain embodiments, the imaging agent is represented byFormula II:M_(m)-[[X]_(r)—X₁*-[X]_(p)—X₂*-[X]_(q)]  (II)wherein:

-   -   X, independently, for each occurrence, is an amino acid residue;    -   X₁* and X₂* are each independently X-L-F;    -   L, independently, for each occurrence, is a linker moiety or a        bond;    -   F is a fluorophore;    -   M is a biological modifier;    -   m is 0, 1 or 2;    -   r is an integer from 0 to 28;    -   p is an integer from 1 to 28;    -   q is an integer from 0 to 28; wherein the sum of r, p and q is        no greater than 28.

Depending upon the circumstances, amino acid residue X₁* is a lysineand/or amino acid residue X₂* is a lysine.

In another aspect, the invention provides an intramolecularly-quenchedimaging agent represented by Formula III:[[ECO-G_(g)]_(p)-M]-K_(n)  (III)wherein:

-   -   ECO is an enzymatically cleavable oligopeptide;    -   G is L-F;    -   F, independently, is selected from a fluorophore or quencher,        wherein at least one F is a fluorophore;    -   L, independently for each occurrence, is selected from a linker        moiety or a bond;    -   M is a biological modifier;    -   K is L-N;    -   N is a non-fluorescent reporter;    -   p is an integer from 1 to 4;    -   n is an integer from 0 to 3; and    -   g, independently, for each occurrence, is an integer from 1 to        2, wherein the sum of each occurrence of g is no greater than 2.

The oligopeptide can comprise from about 2 to about 25 amino acidresidues, from about 2 to about 14 amino acid residues, from about 4 toabout 10 amino acid residues, or from about 5 to about 8 amino acidresidues. In certain embodiments, at least one F is chemically linked,directly or indirectly, to a lysine residue.

In another aspect the invention provides cyclic intramolecularlyquenched imaging agent comprising (a) a first fluorophore chemicallylinked, directly or indirectly, to the C-terminus of a first cleavableoligopeptide and chemically linked, directly or indirectly, to theN-terminus of a second, optionally cleavable, oligopeptide; (b) a secondfluorophore chemically linked, directly or indirectly, to the N-terminusof the first cleavable oligopeptide and chemically linked directly orindirectly, to the C-terminus of the second, optionally cleavableoligopeptide; and (c) optionally, at least one biological modifierchemically linked to the first or second oligopeptide or fluorophore.

In one embodiment, the imaging agent can be represented by Formula IV:

wherein,

-   -   ECO, independently, for each occurrence, is an enzymatically        cleavable oligopeptide;    -   G is L-F-L;    -   F, independently, for each occurrence, is a fluorophore;    -   L, independently, for each occurrence, is a linker moiety or a        bond;    -   M is a biological modifier;    -   K is L-N;    -   N is a non-fluorescent reporter;    -   n is an integer from 0 to 3; and    -   m is an integer from 0 to 3.

In another aspect, the invention provides an intramolecularly-quenchedimaging agent comprising (a) an enzymatically cleavable oligopeptidecomprising from 2 to 14 amino acid residues; (b) at least one biologicalmodifier with a molecular weight of from about 5 kDa to about 35 kDacovalently linked to the enzymatically cleavable oligopeptide; and (c)two fluorophores, each covalently linked, directly or indirectly, to theoligopeptide at locations so that the fluorophores quench one another,and wherein, upon enzymatic cleavage of the oligopeptide, at least onefluorophore becomes unquenched and is capable of emitting a fluorescentsignal when excited by electromagnetic radiation.

In another aspect, the invention provides an intramolecularly-quenchedimaging agent represented by Formula V:

wherein

X₁* independently, at each occurrence, is X-L-;

X is an amino acid residue;

L is a linker moiety or a bond; and

F, independently, at each occurrence, is a fluorophore.

In another aspect, the invention provides an intramolecularly-quenchedimaging agent represented by Formula VI:

wherein

X₁*, independently, at each occurrence, is X-L-;

X is an amino acid residue;

L is a linker moiety or a bond;

F, independently, at each occurrence, is a fluorophore.

In any of the foregoing imaging agents, the fluorophore preferably is afar-red or a near-infrared fluorophore. Exemplary fluorophores includecarbocyanine fluorophore and indocyanine fluorophore. It is understoodthat when the agent comprises two fluorophores, they can be the same ordifferent. Other useful fluorophores and quenchers, as well as usefullinkers, biological modifiers, and non-fluorescent reporters aredescribed in more detail below.

In certain embodiments, the enzymatically cleavable oligopeptide iscleavable by at least one enzyme selected from the group consisting of acathepsin, a matrix metalloprotease, a peptidase, a carboxypeptidase, aglycosidase, a lipase, a phospholipase, a phosphatase, aphosphodiesterase, a sulfatase, a reducatese, and a bacterial enzyme.

In addition, the invention provides methods of imaging in-vivo orin-vitro. In one aspect, the invention provides a method for performingin vivo optical imaging in a subject, for example, a mammal, forexample, an animal or a human. The method comprises the steps of (a)administering to a subject one of more of the foregoing imaging agents,(b) allowing the agent or agents to distribute within the subject; (c)exposing the subject to light of a wavelength absorbable by at least onefluorophore in the agent; and (d) detecting a signal emitted by thefluorophore. The emitted signal can be used to construct an image, forexample, a tomographic image. Furthermore, steps (c)-(d) can be repeatedat predetermined intervals thereby to permit evaluation of the emittedsignals of the agent in the subject over time. Similarly, steps (a)-(d)can be repeated at predetermined time intervals thereby to permitevaluation of the emitted signals of the agent in the subject over time.The presence, absence, or level of emitted signal can be indicative of adisease state and or can be used to detect and/or monitor a disease.

In another aspect, the invention provides an in vitro imaging method.The method comprises (a) contacting a sample, for example, a biologicalsample, with any one or more of the foregoing imaging agents, (b)allowing the agent to bind to or associate with a biological target;optionally, removing unbound agents; and (d) detecting a signal emittedfrom the fluorophore thereby to determine whether the agent has beenactivated by or bound to the biological target.

The imaging agents can also be incorporated into a kit, for example, akit with optional instructions for using the agents during in vivo or invitro imaging. The kit optionally comprises components that aid in theuse of the imaging agents for the disclosed methods, such as buffers,and other formulating agents. Alternatively, the kit can comprisemedical devices that aid in the administration and/or detection of theagents to subjects.

The imaging agents and methods disclosed herein provide variousadvantages and have broad applications in both research and clinicalsettings. For example, the imaging agents and methods permit theacquisition of molecular images and optionally, high resolutionanatomical images. The imaging agents and methods may provide insightsinto specific molecular abnormalities that form the basis of manydiseases, and can be used to assess efficacy of established therapies atthe molecular level. This, in turn, is expected to have an impact indrug development, drug testing, disease diagnosis, and choosingappropriate therapies and therapy changes in a given subject.

Other features and advantages of the invention will be apparent from thefollowing figures, detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a planar image of bilateral tumors at 6 hours afteradministration of an exemplary imaging agent referred to as Q65.

FIG. 2 depicts fluorescence tomography bilateral images of tumors at 6hours after administration of the exemplary imaging agent Q65.

FIG. 3 depicts planar fluorescence reflectance images of 4T1 Xenografttumors using an exemplary agent referred to as Q66.

FIG. 4 depicts a planar fluorescence reflectance image of HT-29Xenograft tumors using an exemplary agent referred to as Q91.

FIG. 5 depicts the enzymatic activation profile of the imaging agentQ65.

FIG. 6 depicts the enzymatic activation profiles of the exemplaryimaging agents, referred to as Q92 (FIG. 6A) and Q93 (FIG. 6B).

FIG. 7A depicts fluorescent images of control (FIGS. 7A and 7C) andovariectomy induced osteoporosis (FIGS. 7B and 7D) in mice taken 4 hours(FIGS. 7A and 7B) and 24 hours (FIGS. 7C and 7D) after administering anexemplary imaging agent referred to as Q94. FIG. 7E is a bar chartshowing quantitation of tibial fluorescence of induced osteoporosis 4hours and 24 hours after administration of imaging agent Q94.

FIG. 8 depicts an image produced by fluorescence reflectance imaging ofcardiovascular disease using the exemplary imaging agent Q65.

FIG. 9 depicts an image of a mouse following carrageenan induced pawedema using the exemplary imaging agent Q91.

FIG. 10 is a schematic representation of an exemplary cyclic imagingagent that comprises two fluorophores separated by two peptides. Eachpeptide contains a proteolytic cleavage site where, when intact, theimaging agent is quenched and when the peptides are cleaved by exposureto one or more proteases, at least one fluorophore is no longer quenchedby the other.

FIG. 11 is a bar chart showing the enzyme activation profile forexemplary imaging agents referred to as R20, R21, R23, R24, R26, andR27.

FIG. 12 shows images of mice on a control or low sodium diet using twoexemplary imaging agents, wherein FIG. 12A represents a mouse on acontrol diet having received the imaging agent R20, FIG. 12B representsa mouse on a low sodium diet having received the imaging agent R20, FIG.12C represents a mouse on a control diet having received the imagingagent R21, and FIG. 12D represents a mouse on a low sodium diet havingreceived the imaging agent R21.

FIG. 13 is a bar chart showing an enzyme activation panel for anexemplary imaging agent referred to as R22.

FIG. 14 show images created by fluorescent reflectance imaging after 6hours (FIG. 14A) and after 24 hours (FIG. 14B) and by tomographicimaging after 6 hours (FIG. 14C) and after 24 hours (FIG. 14D) using theexemplary imaging agent R22.

FIG. 15 is a bar chart showing enzyme activation profiles of exemplaryimaging agents referred to as R51, R52, R53, R55, R56, R57, R58, andR59.

FIG. 16 depicts images and quantification of the images in ApoE −/− andcontrol mice using the exemplary imaging agent R51. FIG. 16A showsimages taken by fluorescent tomography, and FIG. 16B is a bar chartshowing quantification of the resulting images. FIG. 16C is areflectance image, and FIG. 16D is a bar chart showing quantification ofthe resulting images.

FIG. 17 depicts images and quantification of the images in ApoE −/− andcontrol mice using the exemplary imaging agent R55. FIG. 17A showsimages taken by fluorescence tomography, and FIG. 17B is a bar chartshowing quantification of the resulting images.

DETAILED DESCRIPTION

This invention provides imaging agents that include one or morefluorescently labeled peptides. The fluorescence of the imaging agentsis enhanced upon activation, e.g, an increase of fluorescence occurs,due to a cleavage of a peptide sequence by a protease after, before orduring binding of the agent or following internalization of the imagingagent.

For example, the imaging agents of the present invention includes atleast one oligopeptide comprising a proteolytic cleavage site, and twofluorophores or a single fluorophore and a quencher) covalently linked(directly or indirectly) to the oligopeptide or an optional biologicalmodifier such that the fluorescence of at least one fluorophore issignificantly quenched. Cleavage of the peptide by, for example,enzymatic cleavage, the agent emits a fluorescent signal when excited byelectromagnetic radiation of appropriate wavelength and frequency. Incertain embodiments, the imaging agents contain no more than twofluorophores. For example, the imaging agents may contain a fluorescentreporter system consisting of, or consisting essentially of, (i) twofluorophores, (ii) two fluorophores and two quenchers, (iii) twofluorophores and one quencher, or (iv) one fluorophore and one quencher.

As used herein, the term “quench” is understood to mean the process ofpartial or complete reduction of the fluorescent signal from afluorophore. For example, a fluorescent signal can be reducedinter-molecularly or intra-molecularly through the placement of anotherfluorophore (either the same or a different fluorophore) in fluorescentquenching proximity to the first fluorophore or the placement of anon-fluorogenic quenching chromophore molecule (quencher) in fluorescentquenching proximity to the first fluorophore. The agent is de-quenched(or activated), for example, through the enzymatic cleavage of a peptidesequence.

The peptide of the imaging agents optionally can be chemically linked toa biological modifier. Furthermore the imaging agents optionally can bechemically linked to another non-fluorescent reporter. As used herein,the term “chemically linked” is understood to mean connected by anattractive force between atoms strong enough to allow the combinedaggregate to function as a unit. This includes, but is not limited to,chemical bonds such as covalent bonds, non-covalent bonds such as ionicbonds, metallic bonds, and bridge bonds, hydrophobic interactions,hydrogen bonds, and van der Waals interactions.

In one aspect of the invention, the agents of the present invention areintramolecularly-quenched agents that include (a) an enzymaticallycleavable oligopeptide comprising from about 2 to about 30 amino acidresidues; (b) an optional biological modifier chemically linked to theenzymatically cleavable oligopeptide; and (c) either two fluorophores orone fluorophore and one quencher, each covalently linked, directly orindirectly, to the oligopeptide or to the biological modifier, whereinone fluorophore quenches the other fluorophore or the quencher quenchesthe fluorophore and upon enzymatic cleavage of the oligopeptide, atleast one fluorophore becomes unquenched and is capable of producing agreater fluorescent signal when excited by electromagnetic radiationthan before enzymatic cleavage of the oligopeptide.

For example, the imaging agent can comprise one or more oligopeptidesfrom about 2 to about 25 amino acid residues in length, from about 2 toabout 14 amino acid residues in length, from about 5 to about 8 aminoacid residues in length, or from about 4 to about 10 amino acid residuesin length. In certain embodiments, the oligopeptide comprises 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 amino acids.

The biological modifier, before being chemically linked to theenzymatically cleavable oligopeptide, can have a molecular weight lessthan about 35 kDa, for example, from about 10 kDa to about 35 kDa, fromabout 5 kDa to about 30 kDa, or from about 15 kDa to about 25 kDa. Thebiological modifier preferably is linked to the oligopeptide at aposition that does not interfere with fluorescent quenching. Forexample, the biological modifier may be chemically linked to a moiety ofthe oligopeptide that is not positioned between two fluorophores thatquench one another or between a fluorophore and a quencher.

In some embodiments, the imaging agents disclosed herein comprise twofluorophores, which may be the same or different. Imaging agents of theinvention can further comprise a non-fluorescent reporter chemicallylinked to the enzymatically cleavable oligopeptide or the biologicalmodifier.

In one embodiment, the imaging agent is represented by Formula I:

wherein:

-   -   B is [M_(m)-ECO-M_(m)];    -   ECO is the enzymatically cleavable oligopeptide;    -   G is L-F;    -   F is a fluorophore or quencher;    -   M is a biological modifier;    -   K is N-L;    -   N is a non-fluorescent reporter;    -   L, independently, for each occurrence, is a linker moiety or a        bond;    -   n is an integer from 0 to 3 (for example, 0, 1, 2, or 3);    -   m, independently, for each occurrence, is 0 or 1, and optionally        at least one m is 1; and    -   f, independently, for each occurrence, is an integer from 0 to        2, wherein the total number of fluorophores F in the agent is no        greater than 2.

In one embodiment, f is 2 and at least one F is a fluorophore.

Each F may be covalently linked, directly or indirectly (for example,through a linker moiety L), to a different amino acid residue (forexample, a lysine) of the oligopeptide. In another embodiment, at leastone F is covalently linked, directly or indirectly (for example, througha linker moiety L), to the biological modifier M. It is understood thatECO can be a linear oligopeptide, or can be a cyclic oligopeptide.

In another aspect, the imaging agent is represented by formula II:M_(m)-[[X]_(r)—X₁*-[X]_(p)—X₂*-[X]_(q)]  (II)wherein:

-   -   X, independently, for each occurrence, is an amino acid residue;    -   X₁* and X₂* are each independently X-L-F;    -   L, independently, for each occurrence, is a linker moiety or a        bond;    -   F is a fluorophore;    -   M is a biological modifier;    -   m is 0, 1 or 2;    -   r is an integer from 0 to 28;    -   p is an integer from 1 to 28;    -   q is an integer from 0 to 28; wherein the sum of r, p and q is        no greater than 28.

In certain embodiments, the sum of r, p and q is 3, 4, 5, 6, 7, 8, 9 or10. The amino acid residue X₂* and/or X₁* can be, for example, a lysine.

In another aspect, the imaging agent is represented by Formula III:[[ECO-G_(g)]_(p)-M]-K_(n)  (III)wherein:

-   -   ECO is an enzymatically cleavable oligopeptide;    -   G is L-F;    -   F, independently, is selected from a fluorophore or quencher,        wherein at least one F is a fluorophore;    -   L, independently, for each occurrence, is selected from a linker        moiety or a bond;    -   M is a biological modifier;    -   K is L-N;    -   N is a non-fluorescent reporter;    -   p is an integer from 1 to 4;    -   n is an integer from 0 to 3; and    -   g, independently, for each occurrence, is an integer from 1 to        2, wherein the sum of each occurrence of g is no greater than 2.

For example, ECO can be about 5 to about 8 amino acid residues inlength. The integer n can be 0, 1, 2 or 3; g of formula III can be,independently for each occurrence, 1, 2, 3, 4 5 or 6. In certainembodiments, at least one F is chemically linked, directly orindirectly, to a lysine residue of the oligopeptide ECO. For example, F,for each occurrence, is covalently bound to the oligopeptide ECO.

In another aspect, the imaging agent is a cyclic intramolecularlyquenched imaging agent comprising: a) a first fluorophore chemicallylinked, directly or indirectly, to the C-terminus of a first cleavableoligopeptide and chemically linked, directly or indirectly, to theN-terminus of a second, optionally cleavable oligopeptide; b) a secondfluorophore chemically linked, directly or indirectly, to the N-terminusof the first cleavable oligopeptide and chemically linked directly orindirectly, to the C-terminus of the second, optionally cleavable,oligopeptide; and c) optionally, at least one biological modifierchemically linked to the first or second oligopeptide or fluorophore.

In one embodiment, the cyclic agents can be represented by Formula IV:

wherein

-   -   ECO, independently for each occurrence, is an enzymatically        cleavable oligopeptide;    -   G is L-F-L;    -   F, independently, for each occurrence, is a fluorophore;    -   L, independently, for each occurrence, is a linker moiety or a        bond;    -   M is a biological modifier;    -   K is L-N;    -   N is a non-fluorescent reporter;    -   n is an integer from 0 to 3 (for example 0, 1, 2 or 3); and    -   m is an integer from 0 to 3 (for example 0, 1, 2 or 3).

Also contemplated herein are stereoisomeric forms, mixtures ofstereoisomeric forms, and pharmaceutically acceptable salt forms of thedisclosed imaging agents.

I. Fluorophores

As used herein, the term “fluorophore” is understood to mean afluorochrome, a dye molecule, a organic or inorganic fluorophore, ormetal chelate. A fluorophore can include a far-red or a near-infraredfluorophore. In certain embodiments, imaging agents disclosed hereininclude a fluorophore selected from the group consisting of acarbocyanine fluorophore or an indocyanine fluorophore. Exemplaryfluorophores include sulfonated fluorophores. It is understood thatfluorophores can also be nanoparticles having fluorescent or luminescentproperties.

In certain embodiments, the fluorophores are near infrared fluorophores(NIRFs) with absorption and emission maximum between about 600 nm andabout 900 nm. It is appreciated that the use of fluorophores withexcitation and emission wavelengths in other spectrums can also beemployed in the compositions and methods of the present invention.

For example, certain exemplary NIRFs have an extinction coefficient ofat least 30,000 M⁻¹ cm⁻¹ per fluorophore molecule in aqueous medium, orat least 50,000 M⁻¹ cm⁻¹ per fluorophore molecule in aqueous medium.NIRFs preferably also have (1) high quantum yield (i.e., quantum yieldgreater than 5% in aqueous medium), (2) narrow excitation/emissionspectrum, spectrally separated absorption and excitation spectra (i.e.,excitation and emission maxima separated by at least 15 nm), (3) highchemical and photostability, (4) little or no nontoxicity, (5) goodbiocompatibility, biodegradability and excretability, and (6) commercialviability and scalable production for large quantities (i.e., gram andkilogram quantities) required for in vivo and human use.

An extinction coefficient of the agents that include a fluorophore canbe calculated as the ratio of the absorbance of fluorophore at itsabsorption maxima (for example at ˜750 nm for VivoTag-S-750, VisEnMedical) in a 1 cm path length cell to the concentration of particles,(ε=A/cl, where A is absorbance, c is molar concentration and l is pathlength in cm).

In particular, certain carbocyanine or polymethine fluorescentfluorophores can be used to produce the imaging agents of the invention,for example, those described in U.S. Pat. Nos. 6,747,159; 6,448,008;6,136,612; 4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616;5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373;6,043,025; 6,127,134; 6,130,094; 6,133,445; also WO 97/40104, WO99/51702, WO 01/21624, and EP 1 065 250 A1; and Tetrahedron Letters 41,9185-88 (2000).

Various useful, exemplary fluorophores are commercially available andinclude, for example: Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660,AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen);VivoTag680, VivoTag-S680, and VivoTag-S750 (VisEn Medical); Dy677,Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce);HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec);IRDye800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); and ADS780WS,ADS830WS, and ADS832WS (American Dye Source).

Table 1 lists a number of exemplary fluorophores useful in the practiceof the invention together with their spectral properties.

TABLE 1 Absorbance max Fluorophore ε_(max) M⁻¹ cm⁻¹ (nm) Cy5 250,000 649Cy5.5 250,000 675 Cy7 250,000 743 AlexaFlour660 132,000 663AlexaFlour680 184,000 679 AlexaFlour750 280,000 749 VivoTag-S680 220,000674 VivoTag-S750 100,000 750 Dy677 180,000 673 Dy682 140,000 690 Dy752270,000 748 Dy780 170,000 782 DyLight547 150,000 557 DyLight647 250,000653

For example, certain useful fluorophores are represented by thefollowing general Formula VII:

or a salt thereof, wherein:

-   -   X is independently selected from the group consisting of        C(CH₂Y₁)(CH₂Y₂), O, S, and Se;    -   Y₁ and Y₂ are independently selected from the group consisting        of H, C₁-C₂₀ aliphatic group and a C₁-C₂₀ aliphatic group        substituted with —OR*, N(R*)₂ or —SR*;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   R₁ is selected from the group consisting of (CH₂)_(x)CH₃,        (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer        selected from 0 to 6 and n is an integer selected from 2 to 6;    -   R₂ and R₃ independently are selected, for each occurrence, from        the group consisting of H, carboxylate, carboxylic acid,        carboxylic ester, amine, amide, sulfonamide, hydroxyl, alkoxyl,        a sulphonic acid moiety and a sulphonate moiety;    -   R₄ is selected from the group consisting of (CH₂)_(x)CH₃,        (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer        selected from 0 to 6 and n is an integer selected from 2 to 6;        and    -   Q is selected from a group consisting of a heteroaryl ring        substituted with a carboxyl group or 6-membered heteroaryl ring        substituted with a carbonyl group.

In certain embodiments, Q can be selected from the group consisting of(i) a carboxyl functionalized heterocyclic ring, (ii) a carboxylfunctionalized nitrogen containing heterocyclic ring, (iii) a carboxylfunctionalized nitrogen containing 6-membered heterocyclic ring, such aspyridine, pyrimidone, pyrazine, and pyridazine, (iv) a carboxylfunctionalized nitrogen containing 6-membered heterocyclic ring, such aspyridine, (v) a carbonyl functionalized nitrogen containing 6-memberedheterocyclic ring, such as pyridine, (vi) an isonicotinic acid,nicotinic acid and picolinic acid, and a group selected from:

wherein the carboxyl group may also be in the form of an ester, anactivated ester or carbonyl halide that is capable of reacting withnucleophiles, and can be, for example, a CO—O-benzotriazolyl,CO—ON-hydroxysuccinimidyl, CO—O-tetrafluorophenyl,CO—O-pentafluorophenyl, CO—O-imidazole, and CO—O-p-nitrophenyl.

Other useful, exemplary fluorophores are represented by the generalFormula VII:

or a salt thereof, wherein:

X₁ and X₂ are independently selected from the group consisting ofC(CH₂K₁)(CH₂K₂), O, S and Se;

-   -   K₁ and K₂ are independently selected from the group consisting        of H, a C₁-C₂₀ aliphatic group and a C₁-C₂₀ aliphatic group        substituted with —OR*, N(R*)₂ or —SR*;    -   or K₁ and K₂ together are part of a substituted or unsubstituted        carbocyclic, or heterocyclic ring;    -   Y₁ and Y₂ are each independently a benzo-condensed ring, a        naphtha-condensed ring or a pyrido-condensed ring;        -   n₁ is 1, 2, or 3;        -   R₂, R₁₁ and R₁₂ are independently H, F, Br, C₁, C₁-C₆ alkyl,            C₁-C₆ alkoxy, aryloxy, a nitrogen-containing heterocyclic            ring, a nitrogen-containing heteroaromatic ring, a            sulfonate, an iminium ion, or any two adjacent R₁₂ and R₁₁            substituents or R₂ and R₁₁ substituents, when taken in            combination, form a 4-, 5-, or 6-membered substituted or            unsubstituted carbocyclic ring, substituted or unsubstituted            non-aromatic carbocyclic ring or a substituted or            unsubstituted carbocyclic aryl ring, wherein the carbocyclic            rings are each independently optionally substituted one or            more times by C₁-C₆ alkyl, halogen, or OR* or SR*;        -   R₁ and R₁₃ are (CH₂)_(x)CH₃, when x is an integer selected            from 0 to 6; or R₁ and R₁₃ are independently (CH₂)_(n)SO₃ ⁻            or (CH₂)_(n)SO₃H when n is an integer selected from 2 to 6;        -   R₃, R₄ and R₅ are independently selected from the group            consisting of H, carboxylate, carboxylic acid, carboxylic            ester, amine, amide, sulfonamide, hydroxyl, alkoxyl, a            sulphonic acid moiety and a sulphonate moiety;        -   Q is absent, or is selected from a carbonyl moiety or a            substituted or unsubstituted C₁-C₆ alkyl group, wherein 0-2            of the methylene groups of the alkyl group can optionally be            replaced by NH, O or S, or a substituted or unsubstituted            C₁-C₆ carbocyclic, non-aromatic carbocyclic, heterocyclic or            non-aromatic heterocyclic ring wherein the heterocyclic            rings contains 1-2 heteroatoms;        -   R₆ is selected from the group consisting of H, a substituted            or unsubstituted C₁-C₂₀ aliphatic group, a substituted or            unsubstituted aryl, a substituted or unsubstituted            alkylaryl, wherein R₆ is optionally substituted with            halogen, OR*, N(R*)₂ or SR*, when Q is absent, a carbonyl            group, a substituted or unsubstituted C₁-C₆ alkyl group,            wherein 0-2 of the methylene groups of the alkyl group are            replaced by NH, O or S, or a substituted or unsubstituted            C₁-C₆ carbocyclic, non-aromatic carbocyclic, heterocyclic or            non-aromatic heterocyclic ring wherein the heterocyclic            rings contains 1-2 heteroatoms; or        -   R₆ is H, when Q is a carbonyl; and        -   R₇ is selected from the group consisting of H, a substituted            or unsubstituted C₁-C₂₀ aliphatic group, a substituted or            unsubstituted aryl, a substituted or unsubstituted            alkylaryl, wherein R₇ is optionally substituted with            halogen, OR*, N(R*)₂ or SR*; or        -   R₆ and R₇, taken together form a 4-, 5-, 6- or 7-membered            heterocyclic or non-aromatic heterocyclic ring optionally            substituted with halogen, OR*, N(R*)₂ or SR*; or    -   NR₆, Q and CHR₇ together form a substituted or unsubstituted or        heterocyclic or non-aromatic heterocyclic ring system wherein        the rings contain 1 or 2 heteroatoms, wherein rings are        optionally substituted with —OR*, N(R*)₂ or —SR*; and        -   W is absent or is a group selected from the group consisting            of —SO₂NR₆-Q-CHR₇—, —O—, —COO—, and —CONH—;        -   h=0-70; k=0 or 1; d=0-12; m=0-12; p=0-12; and        -   Z is, or contains a N, O or S nucleophile functionality or            is, or contains a functionality capable of reacting with N,            O or S nucleophiles; and

each R* is independently H or C₁₋₂₀ alkyl.

The imaging agents disclosed herein can include various fluorophorederivatives and other forms of fluorophores, such asN-hydroxysuccinimide forms of fluorophores. The fluorophores may bechemically linked to enzymatically cleavable oligopeptides usingchemistries known in the art.

Exemplary fluorophores that can be used in the synthesis of the imagingagents of the invention include, for example, those listed in Table 2.

TABLE 2 No. Fluorophore 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

In another embodiment, the fluorophore is a nanoparticle havingfluorescent or luminescent properties. For example, an enzymaticallycleavable oligopeptide can be grafted onto nanoparticles comprisingsilicon in a form that has fluorescent or luminescent properties orfluorescent nanoparticles. Aggregates of crystalline silicon (asmultiple or single crystals of silicon), porous silicon, or amorphoussilicon, or a combination of these forms, can form the nanoparticle.Preferred fluorescent silicon nanoparticles have a diameter from about0.5 nm to about 25 nm, more preferably from about 2 nm and about 10 nm.The size of nanoparticles can be determined by laser light scattering orby atomic force microscopy or other suitable techniques.

Fluorescent silicon nanoparticles can have excitation and emissionspectra 200 nm to 2000 nm, however, preferred fluorescent siliconnanoparticles have excitation and emission maximum from about 400 nm toabout 1200 nm (and preferably 500 nm-900 nm, for example, 500 nm-600 nm,600 nm-700 nm, 700 nm-800 nm, or 800 nm-900 nm). Preferred fluorescentsilicon nanoparticles also have extinction coefficients of at least50,000 M⁻¹ cm⁻¹ in aqueous medium. Although fluorescent siliconnanoparticles with excitation and emission maximum between 400 nm and1200 nm are preferred, it will be appreciated that the use offluorescent silicon nanoparticles with excitation and emissionwavelengths in other spectrums can also be employed in the compositionsand methods of the present invention. For example, in certainembodiments, the particles can have excitation approximately about300-350 nm, and emission approximately about 400-450 nm.

Fluorescent silicon nanoparticles also have the following properties:(1) high quantum yield (i.e., quantum yield greater than 5% in aqueousmedium), (2) narrow emission spectrum (i.e., less than 75 nm; morepreferably less than 50 nm), (3) spectrally separated absorption andemission spectra (i.e., separated by more than 20 nm; more preferably bymore than 50 nm), (3) have high chemical stability and photostability(i.e., retain luminescent properties after exposure to light), (4) arebiocompatible (as discussed below) or can be made more biocompatible;(5) are non toxic or minimally toxic to cells or subjects at doses usedfor imaging protocols, (as measured for example, by LD₅₀ or irritationstudies, or other similar methods known in the art) and/or (6) havecommercial viability and scalable production for large quantities (i.e.,gram and kilogram quantities) required for in vivo use, for example usein humans.

Fluorescent quantum dots are also contemplated, for example,enzymatically cleavable oligopeptide can be grafted onto a fluorescentquantum dot such as amine T2MP EviTags (Evident Technologies) or QdotNanocrystals (Invitrogen). In general, fluorescent quantum dots arenanocrystals containing several atoms of a semiconductor material(including but not limited to those containing cadmium and selenium,sulfide, or tellurium; zinc sulfide, indium-antimony, lead selenide,gallium arsenide, and silica or ormosil, which have been coated withzinc sulfide to improve the properties of these fluorescent agents.

Fluorophores can include metal oxide nanoparticles that are fluorescentand can be used in a variety of in vitro and vivo applications. In anembodiment, an enzymatically cleavable oligopeptide is conjugated to atleast one fluorescent metal oxide nanoparticle with one or more of thefollowing features: (1) a polymer coating suitable for attaching aplurality of fluorophores thereby achieving large extinctioncoefficients (in excess of 1,000,000 M⁻¹ cm⁻¹), (2) a non-crosslinkedpolymer coating suitable for attaching from about 10 to about 300fluorophores per particle, (3) a polymer coating suitable for attachinga plurality of fluorophores in a manner that does not significantlycompromise the quantum yield of the fluorophores (e.g., thenanoparticles retain at least 50% of the fluorescent signal that iscreated by substantially the same number of free fluorophores whentested under the same conditions), and (4) a polymer coating that isamenable to efficient chemical linking of biomolecules with retention oftheir biological properties to yield molecular imaging agents. Thefluorescent metal oxide nanoparticles can be highly stable molecularimaging agents in vitro, both before and after chemical linking offluorophores and agents, but yet are labile and/or degradable in vivo.

In certain embodiments, one or more different fluorophore molecules canbe covalently linked to the oligopeptide, or alternatively, twosubstantially similar fluorophores can be covalently linked to theoligopeptide, at fluorescene-quenching permissive locations to producethe imaging agents of the present invention.

In certain embodiments, a quencher is used to quench the fluorescentsignal from the fluorophore covalently linked to the oligopeptide. Forexample, an agent can be designed such that the quencher quenches thefluorescence of the fluorophore of the imaging agent when the agent isin an unactivated state, so that the imaging agent exhibits little or nosignal until it is activated. It is understood that the quencher can bea non-fluorescent agent, which when suitably located relative to afluorophore (i.e., at a fluorescence-quenching permissive location) iscapable of quenching the emission signal from the fluorophore. Asdiscussed above, it is understood that certain of the foregoingfluorphores can act to quench the fluorescent signal of another spacedapart fluorophore, when the two fluorophores are positioned atfluorescence-quenching interaction permissive locations.

A number of quenchers are available and known to those skilled in theart including, but not limited to4-{[4-(dimethylamino)-phenyl]-azo}-benzoic acid (DABCYL), QSY®-7(9-[2-[(4-carboxy-1-piperidinyl)sulfonyl]phenyl]-3,6-bis(methylphenylamino)-xanthyliumchloride) (Molecular Probes, Inc., OR), QSY®-33 (Molecular Probes, Inc.,OR), ATTO612Q, ATTO580Q (ATTO-TEC, Germany); Black Hole Quenchers®(Bioresearch Technologies, Novato, Calif.), QXL™680 Acid (AnaSpec, SanJose Calif.), and fluorescence fluorophores such as Cy5 and Cy5.5 (e.g.,2-[5-[3-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benz[e]indol-2-ylidene]-1,3-pentadienyl]-3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benz[e]indolium,inner salt) (Schobel, Bioconjugate 10: 1107, 1999). Other quenchingstrategies can be used, for example, using various solvents to quenchfluorescence of the agents.

Exemplary fluorophores that can quench the emission of otherfluorophores are represented in Table 3.

TABLE 3 No. Quencher 1

2

As with all the imaging agents discussed herein, the two fluorophores orthe fluorophore and the quencher are located within the intact imagingagent at fluorescent-quenching interaction permissive positions. Inother words, a first fluorophore is located close enough in the intactimaging agent to a second fluophore (or quencher) to permit them tointeract photochemically with one another so that the second fluorophore(or quencher) quenches the signal from the first fluorophore. In thecase of the imaging agents with two fluorophores, one fluorophorepreferably quenches the other fluorophore. For principles of quenching,see U.S. Pat. No. 6,592,847.

II. Non-Fluorescent Reporters

The term “non-fluorescent reporter” as used herein, refers to a chemicalmoiety that is not fluorescent but which can be used to provide thecontrast or signal in imaging and is detectable by a non-fluorescentimaging technique. In certain embodiments, other non-fluorescentreporters can be chemically linked with the imaging agents, or can beadministered to a subject simultaneously or sequentially with theimaging agents of the invention. Such reporters can includephotoluminescent nanoparticles, radioisotopes, superparamagnetic agents,X-ray contrast agents, and ultrasound agents. A reporter may alsocomprise therapeutic reporters such as porphyrins, Photofrin®, Lutrin®,Antrin®, aminolevulinic acid, hypericin, benzoporphryrin derivativesused in photodynamic therapy, and radionuclides used for radiotherapy.

(A) Radioactive Reporters

The imaging agents can include one or more radioactive labels.Radioisotopic forms of metals such as copper, gallium, indium,technetium, yttrium, and lutetium can be chemically linked to themetallic imaging agents and can be used for nuclear imaging ortherapeutic applications. Exemplary radioactive labels include, withoutlimitation, ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ⁷⁷Lu, and⁶⁷Cu.

Other exemplary labels include, for example, ¹²³I, ¹²⁴I, ¹²⁵I, ¹¹C, ¹3N,¹⁵O, and ¹⁸F. Other exemplary labels can be therapeuticradiopharmaceuticals including for example, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir.

Chelators or bonding moieties for diagnostic and therapeuticradiopharmaceuticals are also contemplated and can be chemicallyassociated with the imaging agents. Exemplary chelators can be selectedto form stable complexes with radioisotopes that have imagable gamma rayor positron emissions, such as ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, and ⁶⁷Ga.Exemplary chelators include diaminedithiols,monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Chelatorsgenerally are tetradentate with donor atoms selected from nitrogen,oxygen and sulfur, and may include for example, cyclic and acyclicpolyaminocarboxylates such as diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), (DO3A),2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

Chelators or bonding moieties for therapeutic radiopharmaceuticals canbe selected to form stable complexes with the radioisotopes that havealpha particle, beta particle, Auger or Coster-Kronig electronemissions, such as ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁷⁷Lu, and ⁶⁷Cu. Chelators canbe selected from diaminedithiols, monoamine-monoamidedithiols,triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes, andhydrazines, cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA,DO3A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

(B) Magnetic Reporters

Other exemplary reporters can include a chelating agent for magneticresonance agents. Such chelators can include for example,polyamine-polycarboxylate chelators or iminoacetic acid chelators thatcan be chemically linked to the agents.

Chelators for magnetic resonance imaging agents can be selected to formstable complexes with paramagnetic metal ions, such as Gd(III), Dy(III),Fe(III), and Mn(II), are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(met hylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

In one embodiment, the imaging agents are chemically linked tosuperparamagnetic metal oxide nanoparticles that are either (a)non-fluorescent or (b) are fluorescent and can be used in a variety ofin vitro and in vivo applications. Fluorescent metal oxide nanoparticlesthat also have magnetic properties can be used for MRI, thus providing amulti-modality imaging agent.

In certain embodiments, the imaging agents can include a fluorescentand/or non-fluorescent superparamagenetic metal oxide nanoparticle withone or more of the following features: (1) a polymer coating suitablefor attaching a plurality of agents (2) a non-crosslinked polymercoating suitable for attaching from about 10 to about 300 agents perparticle, and (3) a polymer coating that is amenable to efficientchemical linking of the agents with retention of their biologicalproperties to yield molecular imaging agents. The agent modified metaloxide nanoparticle can be a highly stable molecular imaging agent invitro, both before and after chemical linking of the agents, but yet arelabile and/or degradable in vivo.

(C) Ultrasound Reporters

A non-fluorescent reporter can include gas-filled bubbles such asLevovist, Albunex, or Echovist, or particles or metal chelates where themetal ions have atomic numbers 21-29, 42, 44 or 57-83, for example tofacilitate ultrasound imaging. Examples of such compounds are describedin Tyler et al., Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P.Swanson, “Enhancement Agents for Ultrasound: Fundamentals,”Pharmaceuticals in Medical Imaging, pp. 682-87 (1990).

(D) X-Ray Reporters

Exemplary reporters can comprise of iodinated organic molecules orchelates of heavy metal ions of atomic numbers 57 to 83, for example,for X-ray imaging. Examples of such compounds are described in M. Sovak,ed., “Radiocontrast Agents,” Springer-Verlag, pp. 23-125 (1984) and U.S.Pat. No. 4,647,447.

III. Linkers

Linker or spacer moieties can be used to covalently link one or morefluorophores, quenchers, biological modifiers and non-fluorescentreporters to an enzymatically cleavable oligopeptide or to an optionalbiological modifier to produce agents of the present invention. It isunderstood that there is no particular structural, size or contentlimitation of a linker, if present. Linkers can include, for example, avariety of functional groups such as maleimide, dithiopyridyl, thiol,azide, alkene, or alkyne that permit the assembly of molecules ofdiverse architecture.

Linkers can be homofunctional linkers or heterofunctional linkers. Forexample, amine (NH₂)-functionalized moieties can be reacted withbifunctional cross-linkers designed to react with amino groups.Particularly useful conjugation reagents that can facilitate formationof a linker or facilitate covalent linkage between, for example, afluorophore, and an enzymatically cleavable oligopeptide can include aN-hydroxysuccinimide (NHS) ester and/or a maleimide. The NHS ester canreact with the amine group of, for example, a peptide or fluorophore.The maleimide can react with the sulflhydryl group of another molecule.Other particularly useful linker moieties are bifunctional crosslinkerssuch as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), longchain-SPDP, maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS),succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC),succinimidyl iodoacetate (SIA).

In certain embodiments a linker, if present, may be a derivative of adiamine. A diamine moiety or derivative can provide a linker arm ofvarying lengths and chemistries for chemically linking molecules byderivatizing, optionally, with carboxylic acids. Non-limiting examplesof diamines include ethylenediamine (EDA), propylenediamine, spermidine,spermine, hexanediamine, and diamine-amino acids, such as homolysine,lysine, ornithine, diaminobutyric acid and diaminopropionic acid. Inother embodiments, moieties of an imaging agent can be chemically linkedto a dicarboxylic acid, for example, succinic acid, glutaric acid,suberic acid, or adipic acid. In one embodiment, the linker isaminoethylmaleimide.

In certain embodiments, a linker can be formed from an azide moiety thatcan react with substituted alkynes in an azide-acetylene Huisgen[3+2]cycloaddition. In certain embodiments the azide or alkyne linkercan link a polyethyleneglycol (PEG) moiety to, for example, anenzymatically cleavable oligopeptide. Other contemplated linkers includepropargylglycine, pentanoyl, pentynoic acid, propargylic acid, and/orpropargylamine moieties.

In certain embodiments, fluorophores, quenchers, or other reporters aredirectly linked to the imaging agent using reactive NHS ester groups onthe fluorophores, quenchers, or reporters which react with an aminegroup on the enzymatically cleavable oligopeptide. In certain otherembodiments, carboxylic acid groups on the fluorophores, quenchers, orother reporters can be activated in situ by activating agents known inthe art, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate (HBTU),1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-disuccinimidyl carbonate(DSC). In other embodiments, reporters including a sulfhydryl or thiolgroup, can be chemically linked to the agent via a bifunctionalcross-linker that has a second moiety that can react with a sulfhydryl(thiol) group. Such crosslinking agents include, for example and asdescribed above, SPDP, long chain-SPDP, SIA, MBS, SMCC, and others thatare well known in the art.

Useful linker moieties include both natural and non-natural amino acids,oligopeptides, for example, linear or cyclic oligopeptides, and nucleicacids.

The linker can be a peptide or peptide moiety which optionally includesa proteolytic or non-proteolytic cleavage site, such as anester linkage,that can be cleaved due to pH changes at the site of interest.

IV Biological Modifiers

As used herein, the term “biological modifier” is understood to mean anymoiety that can be used to alter the biological properties of theimaging agent, such as, without limitations, making the imaging agentmore water soluble or more dispersible in media for administration,increasing binding specificity, decreasing immunogenicity or toxicity,or modifying pharmacokinetic profile compared to the non-biologicalmodifier modified agents.

In an embodiment, one or more biological modifiers can be chemicallylinked to the enzymatically cleavable oligopeptide or fluorophore. Inone embodiment, the biological modifier is covalently linked to theenzymatically cleavable peptide at a position that is not between twoamino acids covalently linked to a fluorophore or a quencher.

Exemplary biological modifiers include polyethylene glycol (PEG) andderivatives thereof (for example, alkoxy polyethylene glycol (forexample, methoxypolyethylene glycol, ethoxypolyethylene glycol and thelike), branched polypropylene glycol, polypropylene glycol, a graftcopolymer of poly-lysine and methoxypolyethyleneglycol, amino acids,peptides, lipids, fatty acids, palmitate, phospholipids,phospholipid-PEG conjugates, carbohydrates (such as dextran,amino-dextran, carboxymethyl-dextran), iron oxide nanoparticles,sulfonates, polysulfonates, cysteic acid, naphthylalanine,phenylalanine, and 3,3-diphenylpropylamine.

In general, the biological modifier may have a molecular weight of fromabout 2 kDa to less than 50 kDa, such as from about 5 kDa to about 40kDa, such as from about 10 kDa to about 35 kDa, further such as fromabout 15 kDa to 30 kDa. In another embodiment, the biological modifiermay have a molecular weight of from about 5 kDa to about 45 kDa, such asfrom about 5 kDa to about 40 kDa, such as from about 5 kDa to about 35kDa, such as from about 5 kDa to about 30 kDa, such as from about 5 kDato about 25 kDa, such as from about 5 kDa to about 20 kDa, such as fromabout 5 kDa to about 15 kDa, further such as from about 5 kDa to 10 kDa.In another embodiment, the biological modifier may have a molecularweight of from about 2 kDa to less than 50 kDa, such as from about 2 kDato about 45 kDa, such as from about 2 kDa to about 40 kDa, such as fromabout 2 kDa to about 35 kDa, such as from about 2 kDa to about 30 kDa,such as from about 2 kDa to about 25 kDa, such as from about 2 kDa toabout 10 kDa, further such as from about 2 kDa to 5 kDa.

In certain embodiments, as discussed above, the biological modifier maybe a PEG moiety that has a molecular weight, for example, from about 0.5kDa to about 50 kDa, about 5 kDa to about 35 kDa, or about 10 kDa toabout 30 kDa. Alternatively, the PEG may be dPEG, functionalized at adiscrete molecular weight, for example, of about 1100 daltons.

In certain embodiments, the PEG ismethoxyPEG₍₅₀₀₀₎-succinimidylpropionate (mPEG-SPA),methoxyPEG₍₅₀₀₀₎-succinimidylsuccinate (mPEG-SS). Such PEGS arecommercially available from Nektar Therapeutics or SunBiowest orLaysanBio or NOF.

In one embodiment, a PEG moiety may be conjugated to reactive amines onthe enzymatically cleavable oligopeptide or fluorophore via a carboxylfunctionality. Alternatively, the PEG modifier can be conjugated to theenzymatically cleavable oligopeptide or fluorophore by using a thiolreactive cross linker and then reacting with a thiol group on the PEG.

In one embodiment, the PEG may be branched, or Y-shaped, as availablefrom JenKem USA or NOF, or comb-shaped, or synthesized by coupling twoor more PEGs to a small molecule such as glutamic acid.

The omega position of PEG may include a hydroxyl group or a methoxygroup and the PEG may also contain an amino group in the omega position.Such an amino group can in turn be coupled to a variety of agents. Inanother embodiment of the present invention, the biological modifier canbe a pegylated poly-L-lysine or a pegylated poly-D-lysine.

In other embodiments, the biological modifier can bepolyvinylpyrrolidone (PVP)-type polymers. The biological modifier can bea functionalized polyvinylpyrrolidone, for example, carboxy or aminefunctionalized on one (or both) ends of the polymer (as available fromPolymersource) or within the polymer chain.

Alternatively, the biological modifier can include PolyN-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine,carboxy, etc.), Poly(N-isopropyl acrylamide) or functionalizedpoly(N-isopropylacrylamide).

Biological modifiers can include straight or branched chain acyl groups,such as pentynoyl; acidic groups, such as succinyl; lower alkyl groups,such as methyl, ethyl, propyl, etc.; carboxyalkyl groups, such ascarboxyethyl; haloalkyl groups, such as trifluoromethyl; and the like.

In other embodiments, the biological modifier can include, but is notlimited to, proteins, peptides, antibodies and antigen binding fragmentsthereof (for example, Fab, Fab′, (Fab′)₂ fragments), single chainantibodies or sFvs, oligonucleotides, aptamers, glycoproteins, ligandsfor cell receptors, polysaccharides, cell receptors, enzyme substrates,enzyme cofactors, biotin, hormones, neurohormones, neurotransmitters,growth factors, cytokines, lymphokines, lectins, selectins, toxins,nucleic acids, oligonucleotides and derivatives thereof. Otherbiomolecules can also be used, such as folate-mediated targetingmolecules (Leamon & Low, Drug Discovery Today, 6:44-51, 2001),transferrin, vitamins, carbohydrates and ligands that targetinternalizing receptors, including, but not limited to,asialoglycoprotein receptor, somatostatin, nerve growth factor,oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II,atrial natriuretic peptide, insulin, glucagons, prolactin, gonadotropin,various opioids and urokinase-type plasminogen activator. Biomoleculessuch as integrin targeting agents are contemplated, such as α_(v)β₃ andGPα_(IIb)β₃, bombesin, CD4 and VCAM-1. Also contemplated are peptidesfor Hepsin, SPARC, PAR1, colon cancer, Factor 13.

Exemplary peptides for use as biological modifiers include: (Hepsin)Ile-Pro-Leu-Val-Leu-Pro-Leu (SEQ ID NO:1); (SPARC)Ser-Pro-Pro-Thr-Gly-Ile-Asn (SEQ ID NO:2); (VCAM1)Val-His-Pro-Lys-Gln-His-Arg (SEQ ID NO:3); (Cathepsin K)Val-His-Pro-Lys-Gln-His-Arg (SEQ ID NO:4); (E-selection binding peptide)Cys-Asp-Ser-Asp-Ser-Asp-Ile-Thr-Trp-Asp-Gln-Leu-Trp-Asp-Asp-Leu-Met-Lys(SEQ ID NO:5); and (Tat) Arg-Arg-Arg-Arg-Gly-Arg-Arg-Arg-Arg (SEQ IDNO:6).

Other contemplated biological modifiers include membrane, transmembrane,and nuclear translocation signal compounds and sequences, which can bederived from a number of sources including, without limitation, virusesand bacteria. Non-limiting examples include HIV-tat derived peptides,protamine, and polyArg and Arg-rich peptides.

Biological modifiers can also include synthetic compounds including, butnot limited to, small molecule drugs, phototherapeutic molecules andderivatives thereof. Other contemplated biological modifiers includeantibiotics such as vancomycin, clindamycin, chemotherapeutics such asdoxorubicin, molecules such as glycine, derivatives of AMG706, Zactima™,MP-412, erlotinib, sorafenib, dasatinib, lestaurtinib, lapatinib, XL647,XL999, MLN518, PKC412, ST1571, AMN107, AEE788, OSI-930, OSI-817,sunitinib, AG-013736; molecules that target/inhibit VEGF receptors, PDGFreceptor, HER2, SSKI, EphB4, EGFR, FGFR, VEGFR-2, VEGFR-3,serine/threonine and receptor kinases, FLT-3, type III RTKs, c-KIT,Bcr-Abl, CSF-1R, CCR-2, RET, VDGF-2 and photodynamic reagents includingbut not limited to Chlorin e6, Photofrin®, Lutrin®, Antrin®,aminolevulinic acid, hypericin, porphyrins, and porphyrin derivatives,for example, benzoporphyrin derivative.

The biological modifiers may, under certain circumstances, render theimaging agents more useful for biological imaging. For example, thebiological modifier may render agents more water soluble, and/or moredispersible in media for administration and/or may have an increasedbinding specificity, and/or may be less immunogenic and/or less toxic,and/or have a reduced non-specific binding and/or alteredbiodistribution and/or pharmacokinetic profile as compared to anon-biologically modified agents. For example, incorporation ofmethoxypolyethylene glycol (mPEG) or polypeptides may function to modifythe pharmacodynamics and blood clearance rates of the agents in vivo.Other biological modifiers may be chosen to accelerate the clearance ofthe agents from background tissue, such as muscle or liver, and/or fromthe blood, thereby reducing the background interference and improvingimage quality. Additionally, the biological modifiers may also be usedto favor a particular route of excretion, e.g., via the kidneys ratherthan via the liver. The biological modifiers may also aid in formulatingagents in pharmaceutical compositions or may be used to alter orpreserve the signal reporting properties of the agents. In oneembodiment of the present invention, the biological modifier can be apolyamino acid or a peptide, including cyclic peptides. In particular,chemical linking of polyethylene glycol (PEG) or a derivative thereof toagents can result in longer blood residence time (longer circulation)and decreasing immunogenicity.

V. Enzymatically Cleavable Oligopeptides

As used herein, the term “enzymatically cleavable oligopeptide” isunderstood to mean a peptide comprising two or more amino acids (asdefined herein) that are linked by means of a enzymatically cleavablepeptide bond. Also included are moieties that include a pseudopeptide orpeptidomimetic. Examples of cleavable peptide substrates can be found inU.S. Pat. No. 7,439,319.

The term “amino acid” as used herein is understood to mean an organiccompound containing both a basic amino group and an acidic carboxylgroup. Included within this term are natural amino acids (e.g., L-aminoacids), modified and unusual amino acids (e.g., D-amino acids), as wellas amino acids which are known to occur biologically in free or combinedform but usually do not occur in proteins. Natural amino acids include,but are not limited to, alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine,tyrosine, tryptophan, proline, and valine. Other amino acids include,but not limited to, arginosuccinic acid, citrulline, cysteine sulfinicacid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine, ornithine,camitine, selenocysteine, selenomethionine, 3-monoiodotyrosine,3,5-diiodotryosine, 3,5,5′-triiodothyronine, and3,3′,5,5′-tetraiodothyronine.

Modified or unusual amino acids which can be used to practice theinvention include, but are not limited to, D-amino acids, hydroxylysine,dehydroalanine, pyrrolysine, 2-aminoisobutyric acid, gamma aminobutyricacid, 5-hydroxytryptophan, S-adenosyl methionine, S-adenosylhomocysteine, 4-hydroxyproline, an N-Cbz-protected amino acid,2,4-diaminobutyric acid, homoarginine, norleucine, N-methylaminobutyricacid, naphthylalanine, phenylglycine, .beta.-phenylproline,tert-leucine, 4-aminocyclohexylalanine, N-methyl-norleucine,3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine,4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid,trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid,1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoic acid.

As used herein, a “pseudopeptide” or “peptidomimetic” is a compoundwhich mimics the structure of an amino acid residue or a peptide, forexample, by using linking groups other than via amide linkages(pseudopeptide bonds) and/or by using non-amino acid substituents and/ora modified amino acid residue. A “pseudopeptide residue” means thatportion of a pseudopeptide or peptidomimetic that is present in apeptide. The term “pseudopeptide bonds” includes peptide bond isostereswhich may be used in place of or as substitutes for the normal amidelinkage. These substitute or amide “equivalent” linkages are formed fromcombinations of atoms not normally found in peptides or proteins whichmimic the spatial requirements of the amide bond and which shouldstabilize the molecule to enzymatic degradation. The followingconventional three-letter amino acid abbreviations are used herein:Ala=alanine; Aca=aminocaproic acid, Ahx=6-aminohexanoic acid,Arg=arginine; Asn=asparagines; Asp=aspartic acid; Cha=cyclohexylalanine;Cit=citrulline; Cys=cysteine; Dap=diaminopropionic acid; Gln=glutamine;Glu=glutamic acid; Gly=glycine; His=histidine; Ile=isoleucine;Leu=leucine; Lys=lysine; Met=methionine; Nal=naphthylalanine;Nle=norleucine; Om=ornithine; Phe=phenylalanine; Phg=phenylglycine;Pro=praline; Sar=sarcosine; Ser=serine; Thi=Thienylalanine; Thrthreonine; Trp=tryptophan; Tyr=tyrosine; and Val=valine. Use of theprefix D- indicates the D-isomer of that amino acid; for exampleD-lysine is represented as D-Lys.

The peptides can be synthesized using either solution phase chemistry orsolid phase chemistry or a combination of both (Albericio, Curr.Opinion. Cell Biol., 8, 211-221 (2004), M. Bodansky, Peptide Chemistry:A Practical Textbook, Springer-Verlag; N. L. Benoiton, Chemistry ofPeptide Synthesis, 2005, CRC Press).

Selective or orthogonal amine protecting groups may be required toprepare the agents of the invention. As used herein, the term “amineprotecting group” means any group known in the art of organic synthesisfor the protection of amine groups. Such amine protecting groups includethose listed in Greene, “Protective Groups in Organic Synthesis” JohnWiley & Sons, New York (1981) and “The Peptides: Analysis, Synthesis,Biology, Vol. 3, Academic Press, New York (1981). Any amine protectinggroup known in the art can be used. Examples of amine protecting groupsinclude, but are not limited to, the following: 1) acyl types such asformyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromaticcarbamate types such as benzyloxycarbonyl (Cbz or Z) and substitutedbenzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxycarbonyl, and9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate types suchas tert-butyloxycarbonyl (Boc), ethoxycarbonyl,diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkylcarbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl;5) alkyl types such as triphenylmethyl and benzyl; 6) trialkylsilanesuch as trimethylsilane; and 7) thiol containing types such asphenylthiocarbonyl and dithiasuccinoyl. Also included in the term “amineprotecting group” are acyl groups such as azidobenzoyl,p-benzoylbenzoyl, o-benzylbenzoyl, p-acetylbenzoyl, dansyl,glycyl-p-benzoylbenzoyl, phenylbenzoyl, m-benzoylbenzoyl,benzoylbenzoyl.

In certain embodiments the enzymatically cleavable oligopeptide caninclude oligo-L-arginine, oligo-L-lysine, oligo-L-aspartic acid oroligo-L-glutamic acid.

In certain embodiments the enzymatically cleavable oligopeptide includeslysine and arginine. In certain embodiments of the present invention,the enzymatically cleavable oligopeptide can include lysine, arginineand phenylalanine, or may include lysine, phenylalanine and glycine. Inanother embodiment, the enzymatically cleavable oligopeptide can includelysine, phenylalanine, leucine, and glycine, or can include ornithine,phenylalanine, leucine, and glycine. In one embodiment, theenzymatically cleavable oligopeptide may include diaminopropionic acid,ornithine, phenylalanine, leucine, and glycine.

Exemplary enzymatically cleavable oligopeptides are set forth in Table4.

TABLE 4 SEQ ID Oligopeptide NO. Lys-Lys-Lys-Lys-Gly 7 Lys-Lys-Lys-Gly 8Lys-Lys-Gly-Lys-Lys 9 Orn-Lys-Lys-Orn-Gly 10 Orn-Lys-Lys-Orn-Ahx 11Orn-Lys-Lys-Orn-Ahx-Gly-Gly 12 Lys-Lys-Lys-Lys-Ahx 13 Lys-Lys-Lys-βAla14 Lys-Lys-Lys-Lys-Gly-Gly-Gly-Gly-Gly 15 Gly-Gly-Lys-Lys-Lys-Lys-Gly 16Lys-Lys-Lys-Lys-Lys-Lys-Gly 17 D-Lys-Lys-Lys-Lys-D-Lys-Lys-GlyLys-Lys-Lys-Lys-Lys-Lys-Ahx 18Lys-Lys-Lys-Lys-Lys-Lys-Gly-Gly-Gly-Gly-Gly 19Lys-Lys-Lys-Lys-Lys-D-Lys-Gly-Gly-Gly-Gly-GlyLys-Arg-Lys-Arg-Lys-Arg-Gly 20 Lys-Arg-Lys-Arg-Lys-Arg-Gly-Cys 21Lys-Arg-Arg-Arg-Lys-Arg-Gly 22 Lys-Arg-Lys-Arg-Lys-Arg-Lys-Gly 23Lys-Arg-Arg-Arg-Arg-Lys-Arg-Gly 24 Phe-Arg-Lys-Gly-Gly-Arg-Lys 25Phe-Arg-Lys-Gly-Gly-Arg-D-Lys Phe-Arg-Lys-Gly-Gly-Arg-Arg-Lys 26Phe-Arg-Lys-Gly-Gly-Arg-Lys-Ahx 27 Phe-Arg-Lys-Gly-Gly-Arg-Lys-Gly-Gly28 Cha-Arg-Lys-Gly-Gly-Arg-Lys 29 Thi-Arg-Lys-Gly-Gly-Arg-Lys 30Thi-Arg-Lys-Gly-Gly-Arg-Lys-Gly 31 Phe-Gly-Lys-Arg-Arg-Lys 32D-Phe-Gly-Lys-Arg-Arg-Lys Thi-Gly-Lys-Arg-Arg-Lys 33Cha-Gly-Lys-Arg-Arg-Lys 34 Phe-Gly-Orn-Arg-Arg-Dap 35Phe-Gly-Orn-Arg-Arg-Orn 36 Phe-Gly-Lys-Arg-Arg-Lys-Gly-Gly 37Phe-Gly-Lys-Arg-Arg-Lys-Ahx 38 Phe-Gly-Lys-Arg-Arg-Lys-Arg-Arg-Arg-Ahx39 Phe-Gly-Lys-Arg-Arg-Lys-Arg-Arg-Arg-D-Arg-AhxPhe-Gly-Lys-Arg-Arg-Lys-Glu-Glu-Glu-Ahx 40Phe-Gly-Lys-Arg-Arg-Lys-Arg-Arg-Arg-Ahx-Cys 41Lys-Gly-Phe-Leu-Gly-βAla-Lys 42 D-Lys-Gly-Phe-Leu-Gly-βAla-LysOrn-Gly-Phe-Leu-Gly-βAla-Orn 43 Lys-Gly-Phe-Leu-Gly-βAla-Lys-Gly 44Lys-Gly-Phe-Leu-Gly-βAla-Lys-Ahx 45Lys-Gly-Phe-Leu-Gly-βAla-Lys-Gly-Gly-Gly 46Gly-Gly-Gly-Lys-Gly-Phe-Leu-Gly-βAla-Lys 47Ahx-Lys-Gly-Phe-Leu-Gly-βAla-Lys-Ahx 48Lys-Gly-Phe-Leu-Gly-βAla-Lys-Ahx-Cys 49 Gly-Phe-Leu-Gly-Lys 50Lys-Phe-Leu-Gly-Lys-Ahx 51 Gly-Phe-Leu-Gly-Lys-Gly-Gly-Gly 52Gly-Gly-Phe-Leu-Gly-Lys-Ahx 53 Gly-Phe-Leu-Gly-Lys-Cys 54Gly-Phe-Leu-Gly-Lys-Ahx-Cys 55 Gly-Gly-Phe-Leu-Gly-Lys-Ahx-Cys 56Gly-Phe-Leu-Gly-Orn 57 Gly-Phe-Leu-Gly-Lys-Arg-Arg-Arg-Arg-Cys 58His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn-βAla 59His-Gly-Pro-Asn-Orn-His-Gly-Pro-Asn-βAla 60His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn-βAla 61Lys-His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn-βAla 62Orn-His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn-βAla 63Orn-His-Gly-Pro-Asn-Orn-His-Gly-Pro-Asn-βAla 64Phe-Gly-Gly-His-Gly-Pro-Asn-Lys-His-Gly-Pro- 65 Asn-AhxHis-Gly-Pro-Arg-Lys-His-Gly-Pro-Arg-βAla 66His-Gly-Pro-Asn-Lys-His-Gly-Pro-Arg-βAla 67His-Gly-Pro-Arg-Lys-His-Gly-Pro-Asn-βAla 68His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn-Ahx 69His-Gly-Pro-Arg-Lys-His-Gly-Pro-Arg-Gly-Gly- 70 Gly-Phe-GlyHis-Gly-Pro-Arg-Orn-His-Gly-Pro-Arg-βAla 71His-Gly-Pro-Cit-Lys-His-Gly-Pro-Asn-βAla 72His-Gly-Pro-Asn-Lys-His-Gly-Pro-Cit-βAla 73His-Gly-Pro-Asn-Orn-His-Gly-Pro-Cit-βAla 74 His-Gly-Pro-Asn-Lys 75His-Gly-Pro-Asn-Orn 76 Lys-His-Gly-Pro-Asn-Lys 77Orn-His-Gly-Pro-Asn-Lys 78 His-Gly-Pro-Asn-Lys-Gly-Gly-Gly 79His-Gly-Pro-Asn-Lys-Gln-Gly-Gly 80 His-Gly-Pro-Asn-Orn-Gly-Gly-Ahx 81His-Gly-Pro-Asn-Lys-Arg-Arg-Arg-Ahx 82 His-Gly-Pro-Asn-Lys-Arg-Gly-Gly83 Gly-Arg-Arg-Arg-Ahx-Orn-His-Gly-Pro-Asn-Lys- 84 GlyGly-Arg-Arg-Arg-Ahx-D-Lys-His-Gly-Pro-Asn-Lys- Gly His-Gly-Pro-Arg-Lys85 His-Gly-Pro-Arg-Orn 86 Lys-His-Gly-Pro-Arg-Lys 87Orn-His-Gly-Pro-Arg-Lys 88 His-Gly-Pro-Arg-Lys-Ahx 89His-Gly-Pro-Arg-Lys-Gly-Gly-Gly 90 His-Gly-Pro-Arg-Orn-Gly-Gly-Ahx 91His-Gly-Pro-Arg-Lys-Arg-Arg-Arg-Ahx 92 His-Gly-Pro-Arg-Lys-Arg-Gly-Gly93 Gly-Arg-Arg-Arg-Ahx-Lys-His-Gly-Pro-Arg-Lys-Gly 94Gly-Lys-Arg-Arg-Ahx-Orn-His-Gly-Pro-Asn-Orn-Gly 95Gly-Lys-Lys-Arg-Ahx-Orn-His-Gly-Pro-Asn-Orn-Gly 96Gly-Arg-Arg-Arg-Lys-Ahx-His-Gly-Pro-Asn-Lys-Gly 97Lys-Pro-Leu-Gly-Val-Arg-Lys 98 Lys-Gly-Pro-Leu-Gly-Val-Arg-Lys 99Ahx-Lys-Pro-Leu-Gly-Val-Arg-Lys 100 Lys-Pro-Leu-Gly-Val-Arg-Lys-Ahx 101Lys-Pro-Leu-Gly-Val-Arg-Lys-Gln-Ahx 102 Lys-Pro-Leu-Gly-Val-Arg-Orn 103Lys-Pro-Leu-Gly-Val-Arg-Gly-D-Lys Orn-Pro-Leu-Gly-Val-Arg-Orn 104Lys-Pro-Leu-Gly-Val-Arg-Lys-Cys 105 Lys-Gly-Pro-Leu-Gly-Val-Arg-Lys-Cys106 Lys-Pro-Leu-Gly-Val-Arg-Lys-Gly-Gly-Gly 107Lys-Gly-Pro-Leu-Gly-Val-Arg-Lys-Gly-Gly-Gly 108Lys-Pro-Leu-Gly-Val-Arg-Lys-Arg-Arg-Arg 109Lys-Gly-Pro-Leu-Gly-Val-Arg-Lys-Arg-Arg-Arg 110Lys-Val-Arg-Leu-Gly-Pro-Lys 111 Lys-Gly-Val-Arg-Leu-Gly-Pro-Lys 112Ahx-Lys-Val-Arg-Leu-Gly-Pro-Lys 113 Ahx-D-Lys-Val-Arg-Leu-Gly-Pro-D-LysLys-Val-Arg-Leu-Gly-Pro-Lys-Ahx 114 Lys-Val-Arg-Leu-Gly-Pro-Orn 115Orn-Val-Arg-Leu-Gly-Pro-Orn 116 Lys-Val-Arg-Leu-Gly-Pro-Lys-Cys 117Lys-Gly-Val-Arg-Leu-Gly-Pro-Lys-Cys 118Lys-Val-Arg-Leu-Gly-Pro-Lys-Gly-Gly-Gly 119Lys-Gly-Val-Arg-Leu-Gly-Pro-Lys-Gly-Gly-Gly 120Lys-Val-Arg-Leu-Gly-Pro-Lys-Arg-Arg-Arg 121Lys-Val-Arg-Leu-Gly-Pro-Lys-Arg-Arg-Arg-D-ArgLys-Gly-Val-Arg-Leu-Gly-Pro-Lys-Arg-Arg-Arg 122Lys-Gly-His-Pro-Gly-Gly-Pro-Gin-Gly-Lys 123 His-Pro-Gly-Gly-Pro-Gin 124Lys-Gly-His-Pro-Gly-Gly-Pro-Gln-Gly-Orn-Ahx 125Gly-Lys-Gly-His-Pro-Gly-Gly-Pro-Gin-Lys 126Lys-His-Pro-Gly-Gly-Pro-Gin-Lys 127Lys-Gly-His-Pro-Gly-Gly-Pro-Gin-Gly-Lys-Cys 128Lys-Gly-His-Pro-Gly-Gly-Pro-Gin-Lys-Gly-Gly 129Lys-Gly-His-Pro-Gly-Gly-Pro-Gln-Lys-Gly-Gly- 130 Arg-ArgLys-Gly-His-Pro-Gly-Gly-Pro-Gln-Lys-Gly-Arg- 131 Arg-ArgLys-Gly-His-Pro-Gly-Gly-Pro-Gln-Lys-Ahx-Arg- 132 Arg-Arg-Cys-GlyIle-His-Pro-Phe-His-Leu-Val-Ile-His 133Lys-His-Pro-Phe-His-Leu-Val-Ile-His 134Lys-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 135Lys-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 136Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 137Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 138 Thr-Pro-Phe-Ser-Gly-Gln 139Glu-Pro-Phe-Trp-Glu-Asp-Gln 140 Leu-Val-Gly-Gly-Ala 141Tyr-Pro-Gly-Gly-Pro-Gin 142 Val-Ala-Asp-Cys-Ala-Asp-Gln 143Val-Ala-Asp-Cys-Ala-Asp-Arg-Gln 144 Val-Ala-Asp-Cys-Ala-Asp-Asp-Gln 145Val-Ala-Asp-Cys-Arg-Asp-Gln 146 Ala-Pro-Glu-Glu-Ile-Met-Arg-Arg-Gln 147Ala-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln 148Ala-Pro-Glu-Glu-Ile-Met-Pro-Arg-Gln 149 Gly-Phe-Leu-Gly 150Gly-Leu-Phe-Gly 151 Glu-Gly-Phe-Leu-Gly 152 Glu-Lys-Gly-Phe-Leu-Gly-Lys153 Arg-Arg-Glu-Lys-Gly-Phe-Leu-Gly-Lys 154 Arg-Gly-Leu-Gly-Lys 155Gly-Gly-Arg-Arg 156 Gly-Gly Gly-Phe-Cha-Gly 157 Arg-Leu-Val-Gly-Phe-Asp158 Arg-Gly-Phe-Phe-Leu 159 Arg-Gly-Phe-Phe-Pro 160 Ala-Phe-Leu-Gly 161Phe-Pro-Ala-Met 162 Glu-Ala-Ala-Ala 163 Gly-Gly-Arg Gly-Arg Phe-ArgGlu-Lys-Arg-Arg-Lys 164 Succinyl-Glu-Lys-Arg-Arg-Lys 165 Val-Lys-Lys-Arg166 Ala-Pro Ala-Ala-Lys His-Gly-Pro-Asn 167 His-Gly-Pro-Arg 168Gly-Pro-Arg Gly-Pro-Arg-Lys 169 Gly-Pro-Asn Pro-Ala-Gly-Pro 170Asn-Gly-Pro-Asn-Lys 171 His-Gly-Pro-Ile 172 His-Gly-Hyp-Asn 173His-Gly-Pro-Cit 174 His-Gly-hPro-Asn Pro-Leu-Gly-Val-Arg 175Gly-Pro-Leu-Gly-Val-Arg 176 Gly-Pro-Leu-Gly-Val-Arg-Glu 177Gly-Pro-Leu-Gly-Val-Arg-Asp 178 Gly-Pro-Leu-Gly-Met-Arg 179Pro-Leu-Gly-Glu-Arg-Gly 180 Pro-Leu-Gly-Leu-Ala-Gly 181Gly-Val-Arg-Leu-Gly-Pro-Lys 182 Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln 183Val-Pro-Met-Ser-Met-Arg-Gly-Gly 184 Ile-Pro-Val-Ser-Leu-Arg-Ser-Gly 185Arg-Pro-Phe-Ser-Met-Ile-Met-Gly 186 Val-Pro-Leu-Ser-Leu-Thr-Met-Gly 187Val-Pro-Leu-Ser-Leu-Tyr-Ser-Gly 188 Ile-Pro-Glu-Ser-Leu-Arg-Ala-Gly 189Lys-His-Pro-Phe-His-Leu-Val-Ile-His-D-LysLys(COR)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys; 190 R = MeLys(COR)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys; 191 R = CF3Lys(COR)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys; 192 R = EtLys(COR)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys; 193 R = mPEG20 k[R′CO] Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys; 194 R′ = Me[R′CO] Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys; 195 R′ = carboxyethylLys-His-Pro-Phe-His-Leu-Leu-Tyr-His-Lys 196Ile-His-Pro-Phe-His-Leu-Leu-Tyr-His-Lys 197Lys-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Lys 198Ile-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Lys 199Lys-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Lys 200Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Lys 201Lys-His-Pro-Tyr(R)-His-Leu-Val-Ile-His-Lys; 202 R = MeLys-His-Pro-Tyr(R)-His-Leu-Val-Ile-His-Lys; 203 R = EtIle-His-Pro-Tyr(R)-His-Leu-Val-Ile-His-Lys; 204 R = MeIle-His-Pro-Tyr(R)-His-Leu-Val-Ile-His-Lys; 205 R = EtOrn-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 206Dap-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 207Ahx-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 208Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- 209 Ile-His-LysArg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile- 210 His-LysVal-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 211 LysTyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Lys 212Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser- 213 Arg-LysPro-Leu-Ala-Gln-Ala-Val-Lys-Arg-Ser-Ser-Ser- 214 ArgSer-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser- 215 Arg-OrnPro-Leu-Ala-Gln-Ala-Val-Orn-Arg-Ser-Ser-Ser- 216 ArgSer-Pro-Leu-Ala-Asn-Ala-Val-Arg-Ser-Ser-Ser- 217 Arg-LysPro-Leu-Ala-Asn-Ala-Val-Lys-Arg-Ser-Ser-Ser- 218 ArgAla-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln-Lys 219Ala-Pro-Glu-Glu-Ile-Met-Arg-Arg-Gln-Lys 220Ala-Pro-Glu-Glu-Ile-Met-Asp-Gln-Gln-Lys 221Ile-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe 222 Gly-Lys-Asp-Glu-Val-Asp 223Lys-His-Pro-Phe-His-Cha-Val-Ile-His-Lys 451Lys(COR)-His-Pro-Phe-His-Cha-Val-Ile-His-Lys; 452 R = Me, CF3

Other exemplary enzymatically cleavable oligopeptides include aCys-S—S-Cys moiety.

In one embodiment of the present invention, the enzymatically cleavableoligopeptide can be a enzymatically cleavable cyclic peptide, which mayfor example, include about 4 to about 30 amino acids. In one embodimentof the present invention, the agent can include 2-5 cyclic peptides. Inanother embodiment of the present invention, the cyclic peptide mayinclude an acyclic component.

Cyclic peptides can be prepared by methods known in the art, forexample, by the linking the N-terminus of a linear peptide with theC-terminus of the linear peptide: head-to-tail cyclization. Suchlinkages can be via a bond, for example, an amide bond or through alinker. In one embodiment, cyclic peptides can be prepared by thelinking the N-terminus of a linear peptide with the side chaincarboxylic acid of a linear peptide, by the linking the side chain amineof a linear peptide with the C-terminus carboxylic acid of a linearpeptide, or by the linking the side chain amine of a linear peptide withthe side chain carboxylic acid of a linear peptide. For example, cyclicpeptides can be prepared by the linking the side chain thiol group of alinear peptide with the side chain thiol group of a linear peptide; forexample by forming a Cys-Cys disulfide bond. Cyclic peptides can be madein solution phase or on solid phase and that cyclization of linearpeptides can be achieved in solution phase or on solid phase.

Cyclic peptides are described using conventional nomenclature. Forexample a head to tail cyclic peptide containing Arg, Gly, Asp, Phe, Lyscan be written as cyclo(Arg-Gly-Asp-Phe-Lys) (SEQ ID NO:224); a cyclicpeptide containing Cys, Arg, Gly, Asp, Cys can be shown as follows: headto tail: cyclo(Cys-Arg-Gly-Asp-Cys) (SEQ ID NO:225); disulphide:Cys-Arg-Gly-Asp-Cys (SEQ ID NO:225).

In certain embodiments, the enzymatically cleavable cyclic oligopeptidecan include oligo-L-arginine, oligo-L-lysine, oligo-L-aspartic acid oroligo-L-glutamic acid. In certain embodiments the enzymaticallycleavable cyclic oligopeptide includes lysine and arginine. In anotherembodiment of the present invention, the enzymatically cleavable cyclicoligopeptide includes lysine, arginine and phenylalanine. In oneembodiment, an enzymatically cleavable cyclic oligopeptide includeslysine, phenylalanine and glycine, or includes lysine, phenylalanine,leucine, and glycine, or includes ornithine, phenylalanine, leucine, andglycine.

In another embodiment, the enzymatically cleavable cyclic oligopeptidecomprises diaminopropionic acid, ornithine, phenylalanine, leucine, andglycine.

In one embodiment of the present invention, the enzymatically cleavablecyclic oligopeptide may include one or more enzymatically cleavableoligopeptide units.

Exemplary enzymatically cleavable cyclic oligopeptides include thoseshown in Table 5.

TABLE 5 SEQ ID Cyclic Oligopeptide NO. cyclo(Lys-Arg-Arg-Lys-Arg-Arg)226 cyclo(D-Lys-Arg-Arg-Lys-Arg-Arg) cyclo(Lys-Arg-Arg-Arg-Lys-Arg-Arg)227 cyclo(D-Phe-Lys-Arg-Arg-Lys-Arg-Arg-Gly)cyclo(Phe-Lys-Arg-Arg-Phe-Lys-Arg-Arg) 228cyclo(D-Phe-Lys-Arg-Arg-D-PheLys-Arg-Arg)cyclo(Cys-Lys-Arg-Arg-Cys-Lys-Arg-Arg) 229cyclo(Phe-Lys-Arg-Arg-Phe-Lys-Arg-Arg-D-Lys)cyclo(Phe-Lys-Arg-Arg-Phe-Lys-Arg-Arg-Gly-Gly- 230 Gly)cyclo(Lys-His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn- 231 Gly)cyclo(D-Lys-His-Gly-Pro-Asn-D-Lys-His-Gly-Pro- Asn-Gly)cyclo(Ahx-Lys-Arg-Arg-Lys-Arg-Arg) 232cyclo(Lys-Arg-Arg-Lys-Lys-Arg-Arg-Lys) 233cyclo(Gly-Lys-Arg-Arg-Lys-Lys-Arg-Arg-Lys-Gly) 234cyclo(Gly-D-Lys-Arg-Arg-Lys-D-Lys-Arg-Arg-Lys- Gly)

Such cyclopeptides may include peptide structures wherein one or twoproteolytic events may be required for activation of the agent.

The enzymatically cleavable oligopeptide is cleavable by at least oneenzyme chosen from hydrolases, elastases, cathepsins, matrixmetalloproteases, peptidases, exopeptidases, endopeptidases,carboxypeptidases, glycosidases, lipases, nucleases, lyases, amylases,phospholipases, phosphatases, phosphodiesterases, sulfatases, serineproteases, subtilisin, chymotrypsin, trypsin, threonine proteases,cysteine proteases, calpains, papains, caspases, aspartic acidproteases, pepsins, chymosins, glutamic acid proteases, renin,reductases, and parasitic, viral and bacterial enzymes.

VI. Exemplary Imaging Agents

Useful imaging agents can be created using one or more of theenzymatically cleavable oligopeptides, fluorophores, quenchers (ifappropriate), biological modifiers and non-fluorescent reportersdescribed herinabove and coupled together using standard chemistriesknown in the art. The imaging agents can be water soluble or waterdispersible (i.e., sufficiently soluble or suspendable in aqueous orphysiological media solutions). The in vivo half-life of the agent canbe designed to be at least about 10 minutes, but more preferably 30minutes to several hours. The in vivo half-life of the agent preferablyis a time (for example, at least about 30 minutes) sufficient to achievegood tissue exposure, target binding, and imaging signal. In a preferredembodiment, the agent imaging probe is water soluble or dispersible inaqueous media, and is biocompatible i.e., non-toxic having, for example,an LD₅₀ of greater than about 50 mg/kg body weight. The imaging agentsalso preferably do not have any undesired phototoxic properties and/ordisplay low serum protein binding affinity.

In certain embodiments, the disclosed agents include peptide or linkermoieties capable of releasing one, two or more fluorophores from theoligopeptide and/or biological modifier upon contact with an enzyme.Such imaging agents can include a biological modifier that can bereleased from a cleavable oligopeptide by an enzyme that is differentfrom the one that cleaves the peptide.

Certain preferred imaging agents are disclosed in the sections thatfollow.

One exemplary imaging agent represented by Formula Q77 includes[Ac]-Lys(F5)-Gly-Phe-Leu-Gly-Gly-Lys(F5)-[OH] (SEQ ID NO:235), whereinAc is an acetyl group and F5 is represented by:

One exemplary imaging agent represented by Formula Q77 includes

One exemplary imaging agent represented by Formula Q88 includes[Ac]-Lys(F6)-Gly-Phe-Leu-Gly-Gly-Lys(F6)-[OH] (SEQ ID NO:236), whereinAc is an acetyl group and F6 is

One exemplary imaging agent represented by Formula Q88 includes

An exemplary imaging agent represented by Q90 includes[F5]-Gly-Phe-Leu-Gly-Gly-Lys(F5)-[OH] (SEQ ID NO:237), wherein F5 is

An exemplary imaging agent represented by the Formula Q90 can bedepicted as:

Other exemplary imaging agents can include moieties set forth in Table6.

TABLE 6 SEQ ID Imaging Agent NO.[Acetyl]-Lys(F5)-Lys-Lys-Lys(F5)-Lys-Lys-Gly- 238 [OH][Acetyl]-Lys(F5)-Lys-Lys-Lys(F5)-Gly-[OH] 239[F5]-His-Gly-Pro-Asn-Lys(F5)-[OH] 240 [F6]-Gly-Phe-Leu-Gly-Lys(F6)-[OH]241 [F5]-His-Gly-Pro-Arg-Lys(F5)-[OH] 242[F5]-His-Gly-Pro-Asn-Lys(F5)-His-Gly-Pro-Asn- 243 βA-[OH][pentynoyl]-Lys(F5)-His-Pro-Gly-Gly-Pro-Gln- 244 Lys(F5)-[OH][pentynoyl]-Lys(F5)-Gly-His-Pro-Gly-Gly-Pro- 245 Gln-Gly-Lys(F5)-[OH][pentynoyl]-Lys(F5)-Val-Arg-Leu-Gly-Pro- 246 Lys(F5)-[OH][pentynoyl]-Lys(F5)-Pro-Leu-Gly-Val-Arg- 247 Lys(F5)-[OH][pentynoyl]-Phe-Gly-Lys (F5)-Arg-Arg-Lys(F5)- 248 [OH][pentynoyl]-Phe-Arg-Lys(F5)-Gly-Gly-Arg- 249 Lys(F5)-[OH][F5]-Gly-Phe-Leu-Gly-Lys(F5)-[OH] 250Acetyl-Phe-Gly-Lys(F5)-Arg-Arg-Lys(F5)-Gly-[OH] 251Acetyl-Phe-Arg-Lys(F5)-Gly-Gly-Arg-Lys(F5)-[OH] 252cyclo(Lys(F5)-Arg-Arg-Arg-Lys(F5)-Arg-Arg) 253cyclo(Phe-Lys(F6)-Arg-Arg-Phe-Lys(F6)-Arg-Arg- 254 Gly-Gly-Gly)cyclo(Ahx-Lys(F6)-Arg-Arg-Lys(F6)-Arg-Arg) 255[Acetyl]-Lys(F5)-Lys-Lys-Lys(F5)-Lys-Lys-Gly- 256 [mPEG20K][Acetyl]-Lys(F5)-Lys-Lys-Lys(F5)-Gly-[mPEG20K] 257[F5]-His-Gly-Pro-Asn-Lys(F5)-[mPEG20K] 258[F6]-Gly-Phe-Leu-Gly-Lys(F6)-[mPEG20K] 259[F5]-His-Gly-Pro-Arg-Lys(F5)-[mPEG20K] 260[F5]-His-Gly-Pro-Asn-Lys(F5)-His-Gly-Pro-Asn- 261 βA-[mPEG20K][F5]-His-Gly-Pro-Asn-Lys(F5)-[dPEG-1.1k] 262[F5]-His-Gly-Pro-Asn-Lys(F5)-[mPEG-5k] 263[F5]-His-Gly-Pro-Asn-Lys(F5)-[mPEG-10k] 264[pentynoyl]-Lys(F5)-His-Pro-Gly-Gly-Pro-Gln- 265 Lys(F5)-[mPEG20K][pentynoyl]-Lys(F5)-Gly-His-Pro-Gly-Gly-Pro- 266Gln-Gly-Lys(F5)-[mPEG20K] [pentynoyl]-Lys(F5)-Val-Arg-Leu-Gly-Pro- 267Lys(F5)-[mPEG20K] [pentynoyl]-Lys(F5)-Pro-Leu-Gly-Val-Arg- 268Lys(F5)-[mPEG20K] [pentynoyl]-Phe-Gly-Lys (F5)-Arg-Arg-Lys(F5)- 269[mPEG20K] [pentynoyl]-Phe-Arg-Lys-(F5)-Gly-Gly-Arg- 270Lys(F5)-[mPEG20K] [F5]-Gly-Phe-Leu-Gly-Lys(F5)-OH 271[succinyl]-Lys(F5)-Gly-Phe-Leu-Gly-Lys(F5)- 272 [NH₂][mPEG-20k] (F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂] 273[mPEG-40k]-(F5)-Gly-Phe-Leu-Gly-Lys(F5)-[NH₂] 274[diphenylpropylamine]-(F6)-Gly-Phe-Leu-Gly- 275 Lys(F6)-[NH₂][dPEG-1.1k]-(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂] 276[succinyl]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂] 277[mPEG-20k-succinyl]-Lys(F6)-Arg-Arg-Lys(F6)- 278 [NH₂][diphenylpropylamine-succinyl]-Lys(F6)-Arg-Arg- 279 Lys(F6)-[NH₂][dPEG-1.1k-succinyl]-Lys(F6)-Arg-Arg-Lys(F6)- 280 [NH₂][succinyl]-Lys(F6)-Ala-Arg-Arg-Lys(F6)-[NH₂] 281[mPEG-20k]-Lys(F6)-Ala-Arg-Arg-Lys(F6)-[NH₂] 282[succinyl]-Lys(F6)-Arg-Arg-Arg-Lys(F6)-[NH₂] 283[mPEG-20k-succinyl]-Lys(F6)-Ala-Arg-Arg- 284 Lys(F6)-[NH₂][succinyl]-Lys(F5)-Lys-Lys-Lys(F5)-[NH₂] 285[mPEG-20k]-Lys(F5)-Lys-Lys-Lys(F5)-[NH₂] 286[palmitoyl]-Lys(F5)-Phe-Arg-Lys(F5)-[NH₂] 287[Ac]-Lys(F5)-Lys-Lys-Lys(F5)-Gly-[Iron Oxide 288 Nanoparticle][F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[mPEG-20k] 289[F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[mPEG-40k] 290[F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)- 291 [Y-PEG-40k][F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[eda] 292[F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[PVP-6k] 293[F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[Dextran- 294 10k][F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-Glu(mPEG- 295 20k)-[mPEG-20k]Ac-Phe-Gly-Lys(F5)-Arg-Arg-Lys(F5)-Gly- 296 [mPEG20K]Ac-Phe-Arg-Lys(F5)-Gly-Gly-Arg-Lys(F5)- 297 [mPEG20K][F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 298 Lys(F5)-[OH][F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 299 Lys(F5)-[NHR]; R = H[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 300 Lys(F5)-[NHR]; R = Me[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 301 Lys(F5)-[NHR]; R = Et[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 302 Lys(F5)-[NHR]; R = mPEG20k[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 303 Lys(F5)-[NHR]; R = mPEG10k[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 304 Lys(F5)-[NHR]; R = mPEG5k[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 305 Lys(F5)-[NHR]; R = dPEG24[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 306 Lys(F5)-[OH][F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 307 Lys(F5)-[NHR]; R = H[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 308 Lys(F5)-[NHR]; R = Me[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 309 Lys(F5)-[NHR]; R = Et[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 310 Lys(F5)-[NHR]; R = mPEG20k[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 311 Lys(F5)-[NHR]; R = mPEG 10k[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 312 Lys(F5)-[NHR]; R = mPEG5k[F5]Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 313 Lys(F5)-[NHR]; R = dPEG24[F5]Lys (COR)-His-Pro-Phe-His-Leu-Val-Ile-His- 314 Lys(F5)-[OH]; R = Me[F5]Lys (COR)-His-Pro-Phe-His-Leu-Val-Ile-His- 315 Lys(F5)-[OH]; R = CF3[F5]Lys (COMe)-His-Pro-Phe-His-Leu-Val-Ile-His- 316 Lys(F5)-[NHR]; R= mPEG20k [F5]Lys (COCF3)-His-Pro-Phe-His-Leu-Val-Ile- 317His-Lys(F5)-[NHR]; R = mPEG20k [RCO]Lys(F5)-His-Pro-Phe-His-Leu-Val-Ile-His- 318 Lys(F5)-[NH2]; R= carboxyethyl [RCO]Lys (F5)-Ile-His-Pro-Phe-His-Leu-Val-Ile- 319His-Lys(F5)-[NH2]; R = carboxyethyl [RCO]Lys(F5)-His-Pro-Phe-His-Leu-Val-Ile-His- 320 Lys(F5)-[NH2]; R = Me [RCO]Lys(F5)-His-Pro-Phe-His-Leu-Val-Ile-His- 321 Lys(F5)-[NH2]; R = mPEG5k[RCO]Lys (F5)-His-Pro-Phe-His-Leu-Val-Ile-His- 322 Lys(F5)-[NH2]; R= mPEG20k [RCO]Lys (F5)-Ile-His-Pro-Phe-His-Leu-Val-Ile- 323His-Lys(F5)-[NH2]; R = Me [RCO]Lys (F5)-Ile-His-Pro-Phe-His-Leu-Val-Ile-324 His-Lys(F5)-[NH2]; R = mPEG5k [RCO]Lys(F5)-Ile-His-Pro-Phe-His-Leu-Val-Ile- 325 His-Lys(F5)-[NH2]; R = mPEG20k[F5]Orn-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 326 [OH]Orn(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 327 [OH][F5]Orn-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 328 [NHR]; R = H[F5]Orn-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 329 [NHR]; R = Me[F5]Orn-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 330 [NHR]; R = mPEG5k[F5]Orn-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 331 [NHR]; R = mPEG20kOrn(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 332 [NHR] R = HOrn(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 333 [NHR] R = MeOrn(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 334 [NHR] R = mPEG5kOrn(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 335 [NHR] R = mPEG20k[F5]Dap-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 336 [OH]Dap(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 337 [OH][F5]Dap-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 338 [NHR]; R = H[F5]Dap-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 339 [NHR]; R = Me[F5] Dap-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 340 [NHR]; R = mPEG5k[F5]Dap-His-Pro-Phe-His-Leu-Val-Ile-His- 341 Lys(F5)[NHR]; R = mPEG20kDap(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 342 [NHR] R = HDap(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 343 [NHR] R = MeDap(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 344 [NHR] R = mPEG5kDap(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 345 [NHR] R = mPEG20k[F5] Ahx-His-Pro-Phe-His-Leu-Val-Ile-His- 346 Lys(F5)[OH]Ahx(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 347 [OH][F5]Ahx-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 348 [NHR]; R = H[F5]Ahx-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 349 [NHR]; R = Me[F5]Ahx-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 350 [NHR]; R = mPEG5k[F5]Ahx-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 351 [NHR]; R = mPEG20kAhx(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 352 [NHR]; R = HAhx(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 353 [NHR]; R = MeAhx(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 354 [NHR]; R = mPEG5kAhx(F5)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5) 355 [NHR]; R = mPEG20k[F5]Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu- 356 Val-Ile-His-Lys(F5)[OH][F5]Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu- 357Val-Ile-His-Lys(F5)[NHR]; R = H[F5]Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu- 358Val-Ile-His-Lys(F5)[NHR]; R = Me[F5]Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu- 359Val-Ile-His-Lys(F5)[NHR]; R = mPEG5k[F5]Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu- 360Val-Ile-His-Lys(F5)[NHR]; R = mPEG20k[F5]Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- 361 Ile-His-Lys(F5)[OH][F5]Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- 362 Ile-His-Lys(F5)[NHR]; R= H [F5]Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- 363Ile-His-Lys(F5)[NHR]; R = Me[F5]Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- 364 Ile-His-Lys(F5)[NHR]; RmPEG5k [F5]Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- 365Ile-His-Lys(F5)[NHR]; R mPEG20k[F5]Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile- 366 His-Lys(F5)[OH][F5]Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile- 367 His-Lys(F5)[NHR]; R = H[F5]Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile- 368 His-Lys(F5)[NHR]; R= Me [F5]Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile- 369 His-Lys(F5)[NHR];R = mPEG5k [F5]Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile- 370His-Lys(F5)[NHR]; R = mPEG20k[F5]Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 371 Lys(F5)[OH][F5]Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 372 Lys(F5)[NHR]; R = H[F5]Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 373 Lys(F5)[NHR]; R = Me[F5]Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 374 Lys(F5)[NHR]; R= mPEG5k [F5]Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His- 375 Lys(F5)[NHR];R = mPEG20k [F5]Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser- 376Ser-Arg-Lys(F5) [F5]Pro-Leu-Ala-Gln-Ala-Val-Lys(F5)-Arg-Ser- 377Ser-Ser-Arg [F5]Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser- 378Ser-Arg-Orn(F5) [F5]Pro-Leu-Ala-Gln-Ala-Val-Orn(F5)-Arg-Ser- 379Ser-Ser-Arg [F5]Ser-Pro-Leu-Ala-Asn-Ala-Val-Arg-Ser-Ser- 380Ser-Arg-Lys(F5) [F5]Pro-Leu-Ala-Asn-Ala-Val-Lys(F5)-Arg-Ser- 381Ser-Ser-Arg [F5]Ala-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln-Lys(F5) 382[F5]Ala-Pro-Glu-Glu-Ile-Met-Arg-Arg-Gln-Lys 383[F5]Ala-Pro-Glu-Glu-Ile-Met-Asp-Gln-Gln-Lys 384[F5]Lys-His-Pro-Phe-His-Cha-Val-Ile-His-Lys(F5) 453 [OH][F5]Lys-His-Pro-Phe-His-Cha-Val-Ile-His-Lys(F5) 454 [NHR]; R = mPEG5k[F5]Lys-His-Pro-Phe-His-Cha-Val-Ile-His-Lys(F5) 455 [NHR]; R = mPEG20k[F5]Lys(COR)-His-Pro-Phe-His-Cha-Val-Ile-His- 456 Lys(F5)[OH]; R = Me[F5]Lys(COR)-His-Pro-Phe-His-Cha-Val-Ile-His- 457 Lys(F5)[OH]; R = CF3

As used in Table 6, F5 is the fluorochrome as depicted above in agentQ77, F6 is the fluorochrome as depicted above in agent Q88, MPEG ismethoxypolyethylene glycol of a specified molecular weight (for example,mPEG20K is a 20 kDa methoxypolyethylene glycol). Ahx is aminohexanoicacid, Orn is ornithine, and DAP is 2,3-diaminopropionic acid.

FIG. 10 is a schematic representation of an exemplary cyclic imagingagent. The cyclic imaging agent comprises two peptides connected at eachend to a fluorophore. A first peptide has a first fluorophore attachedin the vicinity of its N-terminus and a second fluorophore attached inthe vicinity of its C-terminus, and a second oligopeptide has the firstfluorophore attached in the vicinity of its C-terminus and the secondfluorophore attached in the vicinity of its N-terminus. When intact, thefluorophore quenches the other fluorophore, and more preferably eachfluorophore quenches the other fluorophore. It is understood, however,that one of the fluorophores could be replaced with a suitable nonfluorescent quencher. Although both peptides, as shown, contain aproteolytic cleavage site, it is understood that, under certaincircumstances, only one of the two peptides may have a proteolyticcleavage site. Upon cleavage of the proteolytic cleavage site(s), thefluorophores are no longer constrained in fluorescent quenchingproximity to one another and the fluorophores can now fluorescence uponexcitation following exposure to light of an appropriate wavelength.

Exemplary cyclic imaging agents can include an enzymatically cleavablecyclic oligopeptide, for example, an agent represented by Formula IX,wherein, for each occurrence, X is an amino acid residue, M is abiological modifier, F is a fluorophore or quencher, X₁* is X-L, X₂* isX-L, and L is a linker moiety or a bond:

Another exemplary enzymatically cleavable cyclic oligopeptide isrepresented by Formula X, wherein each X, M, F, X₁* and X₂* is asdefined for Formula IX:

In another embodiment, an enzymatically cleavable cyclic oligopeptide isrepresented by Formula XI, wherein each X, M, F, X₁* and X₂* is asdefined in Formula II, above:

Other exemplary enzymatically cleavable cyclic oligopeptides arerepresented by Formulas V, VI, and XII-XVII wherein, each F, M, X₁*, andX₂* is as defined in Formula IX:

An enzymatically cleavable cyclic oligopeptide is represented by FormulaXIII, wherein the fluorophore and modifiers are as discussedhereinabove:

In an exemplary embodiment, an enzymatically cleavable cyclicoligopeptide imaging agent can also be represented as Formula XIXwherein the biological modifiers are as described hereinabove:

The resulting imaging agents preferably have a molecular weight fromabout 2 kDa to about 60 kDa, such as from about 5 kDa to about 50 kDa,such as from about 10 kDa to about 40 kDa, or from about 20 kDa to about30 kDa. In certain embodiments, the imaging agent may have a molecularweight of from about 2 kDa to about 50 kDa, such as from about 2 kDa toabout 45 kDa, such as from about 2 kDa to about 40 kDa, such as fromabout 2 kDa to about 35 kDa, such as from about 2 kDa to about 30 kDa,such as from about 2 kDa to about 25 kDa, such as from about 2 kDa toabout 10 kDa, or such as from about 2 kDa to 5 kDa. In certain otherembodiments, the intact imaging agent may have a molecular weight offrom about 5 kDa to about 50 kDa, such as from about 5 kDa to about 45kDa, such as from about 5 kDa to about 40 kDa, such as from about 5 kDato about 35 kDa, such as from about 5 kDa to about 30 kDa, such as fromabout 5 kDa to about 25 kDa, such as from about 5 kDa to about 20 kDa,such as from about 5 kDa to about 15 kDa, or such as from about 5 kDa toabout 10 kDa.

VII. Formulations

The imaging agents disclosed herein can be formulated into apharmaceutical composition suitable for administration to a subject, forexample, an animal and/or a human. The pharmaceutical composition caninclude one or more imaging agents and one or more excipients, forexample, a stabilizer in a physiologically relevant carrier.

For in vivo use, the compositions of the present invention can beprovided in a formulation suitable for administration to a subject, forexample, an animal or a human. Accordingly, the formulations include theagents together with a physiologically relevant carrier suitable for thedesired form and/or dose of administration. The term, “physiologicallyrelevant carrier” is understood to mean a carrier in which the agentsare dispersed, dissolved, suspended, admixed and physiologicallytolerable, i.e., can be administered to, in, or on the subject's bodywithout undue discomfort, or irritation, or toxicity. The preferredcarrier is a fluid, preferably a liquid, more preferably an aqueoussolution; however, carriers for solid formulations, topicalformulations, inhaled formulations, ophthalmic formulations, andtransdermal formulations are also contemplated as within the scope ofthe invention.

It is contemplated that the agents can be administered orally orparenterally. For parenteral administration, the agents can beadministered intravenously, intramuscularly, cutaneously,percutaneously, subcutaneously, rectally, nasally, vaginally, andocularly. Thus, the composition may be in the form of, e.g., solidtablets, capsules, pills, powders including lyophilized powders,colloidal suspensions, microspheres, liposomes granulates, suspensions,emulsions, solutions, gels, including hydrogels, pastes, ointments,creams, plasters, irrigation solutions, drenches, osmotic deliverydevices, suppositories, enemas, injectables, implants, sprays, oraerosols. The pharmaceutical compositions can be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, 20th edition, 2000, ed. A. R. Germaro,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

It is understood that the formulation of the agents, the choice of modeof administration, the dosages of agents administered to the subject,and the timing between administration of the agents and imaging iswithin the level of skill in the art.

VIII. Imaging Methods

The present invention provides methods for in vitro and in vivo imagingusing the imaging agents disclosed herein. For a review of opticalimaging techniques, see, e.g., Alfano et al., Ann. NY Acad. Sci.820:248-270 (1997); Weissleder, Nature Biotechnology 19, 316-317 (2001);Ntziachristos et al., Eur. Radiol. 13:195-208 (2003); Graves et al.,Curr. Mol. Med. 4:419-430 (2004); Citrin et al., Expert Rev. AnticancerTher. 4:857-864 (2004); Ntziachristos, Ann. Rev. Biomed. Eng. 8:1-33(2006); Koo et al., Cell Oncol. 28:127-139 (2006); and Rao et al., Curr.Opin. Biotechnol. 18:17-25 (2007).

Optical imaging includes all methods from direct visualization withoutuse of any device and use of devices such as various scopes, cathetersand optical imaging equipment, for example computer based hardware fortomographic presentations. The imaging agents are useful with opticalimaging modalities and measurement techniques including, but not limitedto: endoscopy; fluorescence endoscopy; luminescence imaging; timeresolved transmittance imaging; transmittance imaging; nonlinearmicroscopy; confocal imaging; acousto-optical imaging; photoacousticimaging; reflectance spectroscopy; spectroscopy; coherenceinterferometry; interferometry; optical coherence tomography; diffuseoptical tomography and fluorescence mediated molecular tomography(continuous wave, time domain frequency domain systems and earlyphoton), and measurement of light scattering, absorption, polarization,luminescence, fluorescence lifetime, quantum yield, and quenching.

An imaging system useful in the practice of the invention typicallyincludes three basic components: (1) an appropriate light source forinducing excitation of the imaging agent, (2) a system for separating ordistinguishing emissions from light used for fluorophore excitation, and(3) a detection system. The detection system can be hand-held orincorporated into other useful imaging devices, such as intraoperativemicroscopes. Exemplary detection systems include an endoscope, catheter,tomographic system, hand-held imaging system, or a intraoperativemicroscope.

Preferably, the light source provides monochromatic (or substantiallymonochromatic) light. The light source can be a suitably filtered whitelight, i.e., bandpass light from a broadband source. For example, lightfrom a 150-watt halogen lamp can be passed through a suitable bandpassfilter commercially available from Omega Optical (Brattleboro, Vt.).Depending upon the system, the light source can be a laser. See, e.g.,Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994;Ntziachristos et al., Proc. Natl. Acad. Sci. USA 97:2767-2772, 2000; andAlexander, J. Clin. Laser Med. Surg. 9:416-418, 1991. Information onlasers for imaging can be found, for example, at Imaging DiagnosticSystems, Inc., Plantation, Fla. and various other sources. A high passor bandpass filter can be used to separate optical emissions fromexcitation light. A suitable high pass or bandpass filter iscommercially available from Omega Optical, Burlington, Vt.

In general, the light detection system can be viewed as including alight gathering/image forming component and a light/signaldetection/image recording component. Although the light detection systemcan be a single integrated device that incorporates both components, thelight gathering/image forming component and light detection/imagerecording component are discussed separately.

A particularly useful light gathering/image forming component is anendoscope. Endoscopic devices and techniques which have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. Photochem. Photobiol. B 52:131-135, 1999),ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132, 1997), colonand rectum (Mycek et al., Gastrointest. Endosc. 48:390-394, 1998; andStepp et al., Endoscopy 30:379-386, 1998), bile ducts (Izuishi et al.,Hepatogastroenterology 46:804-807, 1999), stomach (Abe et al., Endoscopy32:281-286, 2000), bladder (Kriegmair et al., Urol. Int. 63:27-31, 1999;and Riedl et al., J. Endourol. 13:755-759, 1999), lung (Hirsch et al.,Clin Cancer Res 7:5-220, 2001), brain (Ward, J. Laser Appl. 10:224-228,1998), esophagus, and head and neck regions can be employed in thepractice of the present invention.

Other types of light gathering components are catheter-based devices,including fiber optics devices. Such devices are particularly suitablefor intravascular imaging. See, e.g., Teamey et al., Science276:2037-2039, 1997; and Circulation 94:3013, 1996.

Still other imaging technologies, including phased array technology(Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994; Chance,Ann. NY Acad. Sci. 838:29-45, 1998), optical tomography (Cheng et al.,Optics Express 3:118-123, 1998; and Siegel et al., Optics Express4:287-298, 1999), intravital microscopy (Dellian et al., Br. J. Cancer82:1513-1518, 2000; Monsky et al., Cancer Res. 59:4129-4135, 1999; andFukumura et al., Cell 94:715-725, 1998), confocal imaging (Korlach etal., Proc. Natl. Acad. Sci. USA 96:8461-8466, 1999; Rajadhyaksha et al.,J. Invest. Dermatol. 104:946-952, 1995; and Gonzalez et al., J. Med.30:337-356, 1999) and fluorescence molecular tomography (FMT)(Nziachristos et al., Nature Medicine 8:757-760, 2002; U.S. Pat. No.6,615,063, PCT WO 03/102558, and PCT WO 03/079015) can be used with theimaging agents of the invention. Similarly, the imaging agents can beused in a variety of imaging systems, for example, (1) the IVIS®ImagingSystems: 100 Series, 200 Series (Xenogen, Alameda, Calif.), (2) SPECTRUMand LUMINA (Xenogen, Alameda, Calif.), (3) the SoftScan® or the eXploreOptix™ (GE Healthcare, United Kingdom), (4) Maestro™ and Nuance™-2Systems (CR1, Woburn, Mass.), (5) Image Station In-Vivo FX fromCarestream Molecular Imaging, Rochester, N.Y. (formerly Kodak MolecularImaging Systems), (6) OV100, IV100 (Olympus Corporation, Japan), (7)Cellvizio Mauna Kea Technologies, France), (8)] NanoSPECT/CT or HiSPECT(Bioscan, Washington, D.C.), (9) CTLM® or LILA™ (Imaging DiagnosticSystems, Plantation, Fla.), (10) DYNOT™ (NIRx Medical Technologies, GlenHead, N.Y.), and (11) NightOWL Imaging Systems by Berthold Technologies,Germany.

A variety of light detection/image recording components, e.g., chargecoupled device (CCD) systems or photographic film, can be used in suchsystems. The choice of light detection/image recording depends onfactors including the type of light gathering/image forming componentbeing used. It is understood, however, that the selection of suitablecomponents, assembling them into an optical imaging system, andoperating the system is within ordinary skill in the art.

For agents that have magnetic properties, MRI imaging well known in theart can also be applied in the practice of the invention. For a reviewof MRI techniques see Westbrook, Handbook of MRI Technique, 2^(nd)Edition, 1999, Blackwell Science. It is possible that images obtained,for example, by optical imaging and by magnetic resonance imaging can beco-registered or fused with one another to provide additionalinformation about the item being imaged. Furthermore, multi-modalityimaging systems (i.e., combined optical and MR imaging systems) can beused to create combined optical MR images.

In addition, the compositions and methods of the present invention canbe used for other imaging compositions and methods. For example, theagents of the present invention can be imaged by other imagingmodalities, such as, X-ray, computed tomography (CT), MR imaging,ultrasound, positron emission tomography (PET), and single photoncomputerized tomography (SPECT).

In addition, the compositions and methods of the present invention canbe used in combination with other imaging compositions and methods. Forexample, the agents of the present invention can be imaged by opticalimaging protocols either alone or in combination with other traditionalimaging modalities, such as, X-ray, computed tomography (CT), MRimaging, ultrasound, positron emission tomography (PET), and singlephoton computerized tomography (SPECT). For instance, the compositionsand methods of the present invention can be used in combination with CTor MRI to obtain both anatomical and molecular informationsimultaneously, for example, by co-registration of with an imagegenerated by another imaging modality. The compositions and methods ofthe present invention can also be used in combination with X-ray, CT,PET, ultrasound, SPECT and other optical and MR contrast agents oralternatively, the agents of the present invention may also includeimaging agents, such as iodine, gadolinium atoms and radioactiveisotopes, which can be detected using CT, PET, SPECT, and MR imagingmodalities in combination with optical imaging. The imaging agents canbe linked to or incorporated in the agents.

(A) In Vivo Imaging

With respect to optical in vivo imaging, such a method comprises (a)administering to a subject one or more imaging agents; (b) allowing theagent(s) to distribute within the subject; (c) exposing the subject tolight of a wavelength absorbable by at least one fluorophore in theimaging agent; and (d) detecting an optical signal emitted by thefluorophore. The emitted optical signal can be used to construct animage. The image can be a tomographic image. Furthermore, it isunderstood that steps (a)-(d) or steps (c)-(d) can be repeated atpredetermined intervals thereby to permit evaluation of the subject overtime.

The illuminating and/or detecting steps (steps (c) and (d),respectively) can be performed using an endoscope, catheter, tomographicsystem, planar system, hand-held imaging system, goggles, or anintraoperative imaging system or microscope.

Before or during these steps, a detection system can be positionedaround or in the vicinity of a subject (for example, an animal or ahuman) to detect signals emitted from the subject. The emitted signalscan be processed to construct an image, for example, a tomographicimage. In addition, the processed signals can be displayed as imageseither alone or as fused (combined) images.

In addition, it is possible to practice an in vivo imaging method thatselectively detects and images one or more molecular imaging probes,including the imaging agents simultaneously. In such an approach, forexample, in step (a) noted above, two or more imaging probes whosesignal properties are distinguishable from one another are administeredto the subject, either at the same time or sequentially, wherein atleast one of the molecular imaging probes is a agent. The use ofmultiple probes permits the recording of multiple biological processes,functions or targets.

The subject may be a vertebrate, for example, a mammal, for example, ahuman. The subject may also be a non-vertebrate (for example, C.elegans, drosophila, or another model research organism, etc.) used inlaboratory research.

Information provided by such in vivo imaging approaches, for example,the presence, absence, or level of emitted signal can be used to detectand/or monitor a disease in the subject. Exemplary diseases include,without limitation, autoimmune disease, bone disease, cancer,cardiovascular disease, environmental disease, dermatological disease,immunologic disease, inherited disease, infectious disease, metabolicdisease, neurodegenerative disease, ophthalmic disease, and respiratorydisease. In addition, in vivo imaging can be used to assess the effectof a compound or therapy by using the imaging agents, wherein thesubject is imaged prior to and after treatment with the compound ortherapy, and the corresponding signal/images are compared.

The invention also features an in vivo imaging method where labeledcells are administered to the recipient. The cells can be labeled withthe imaging agents ex vivo. The cells can be derived directly from asubject or from another source (e.g., from another subject, cellculture, etc.). The imaging agents can be mixed with the cells toeffectively label the cells and the resulting labeled cells administeredto the subject into a subject in step (a). Steps (b)-(d) then arefollowed as described above. This method can be used for monitoringtrafficking and localization of certain cell types, including T-cells,tumor cells, immune cells and stem cells, and other cell types. Inparticular, this method may be used to monitor cell-based therapies.

The methods of the invention can be used to determine a number ofindicia, including tracking the localization of the agent in the subjectover time or assessing changes or alterations in the metabolism and/orexcretion of the agent in the subject over time. The methods can also beused to follow therapy for such diseases by imaging molecular events andbiological pathways modulated by such therapy, including but not limitedto determining efficacy, optimal timing, optimal dosing levels(including for individual patients or test subjects), and synergisticeffects of combinations of therapy.

The methods and compositions described herein can be used to help aphysician or surgeon to identify and characterize areas of disease, suchas arthritis, cancers and specifically colon polyps, or vulnerable orunstable plaque, atherosclerosis, to distinguish diseased and normaltissue, such as detecting tumor margins that are difficult to detectusing an ordinary operating microscope, e.g., in brain surgery, to helpdictate a therapeutic or surgical intervention, e.g., by determiningwhether a lesion is cancerous and should be removed or non-cancerous andleft alone, or in surgically staging a disease, e.g., intraoperativelymph node staging, sentinel lymph node mapping, or assessingintraoperative bleeding.

The methods and compositions described herein can also be used in thedetection, characterization and/or determination of the localization ofa disease, especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state.

The methods and compositions disclosed herein can also be used tomonitor and/or guide various therapeutic interventions, such as surgicalprocedures, and monitoring drug therapy, including cell based therapies.The methods can also be used in prognosis of a disease or diseasecondition.

With respect to each of the foregoing, examples of such disease ordisease conditions that can be detected or monitored (before, during orafter therapy) include inflammation (for example, inflammation caused byarthritis, for example, rheumatoid arthritis), cancer (for example,colorectal, ovarian, lung, breast, prostate, cervical, testicular, skin,brain, gastrointestinal, pancreatic, liver, kidney, bladder, stomach,leukemia, mouth, esophageal, bone), cardiovascular disease (for example,atherosclerosis and inflammatory conditions of blood vessels, ischemia,hypertension, stroke, thrombosis, disseminated intravascularcoagulation), dermatologic disease (for example, Kaposi's Sarcoma,psoriasis, allergic dermatitis), ophthalmic disease (for example,macular degeneration, diabetic retinopathy), infectious disease (forexample, bacterial, viral, fungal and parasitic infections, includingAcquired Immunodeficiency Syndrome, Malaria, Chagas Disease,Schistosomiasis), immunologic disease (for example, an autoimmunedisorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetesmellitus, lupus erythematosis, myasthenia gravis, Graves disease),central nervous system disease (for example, a neurodegenerativedisease, such as Parkinson's disease or Alzheimer's disease,Huntington's Disease, amyotrophic lateral sclerosis, prion disease),inherited diseases, metabolic diseases, environmental diseases (forexample, lead, mercury and radioactive poisoning, skin cancer),bone-related disease (for example, osteoporosis, primary and metastaticbone tumors, osteoarthritis), neurodegenerative disease, andsurgery-related complications (such as graft rejection, organ rejection,alterations in wound healing, fibrosis or other complications related tosurgical implants).

The methods and compositions described herein, therefore, can be used,for example, to determine the presence and/or localization of tumorcells, the presence and/or localization of inflammation, including thepresence of activated macrophages, for instance in atherosclerosis orarthritis, the presence and in localization of vascular diseaseincluding areas at risk for acute occlusion (i.e., vulnerable plaques)in coronary and peripheral arteries, regions of expanding aneurysms,unstable plaque in carotid arteries, and ischemic areas. The methods andcompositions of the invention can also be used in identification andevaluation of cell death, injury, apoptosis, necrosis, hypoxia andangiogenesis. The methods and compositions can also be used for drugdelivery and to monitor drug delivery, especially when drugs ordrug-like molecules are chemically attached to the imaging agents.

(B) In Vitro Methods

With respect to in vitro imaging, the imaging agents can be used in avariety of in vitro assays. For example, an exemplary in vitro imagingmethod comprises: (a) contacting a sample, for example, a biologicalsample, with one or more imaging agents of the invention; (b) allowingthe agent(s) to interact with a biological target in the sample; (c)optionally, removing unbound agents; (d) in the case of fluorescentagents, illuminating the sample with light of a wavelength absorbable bya fluorophore of the agents; and (e) detecting a signal emitted fromfluorophore thereby to determine whether the agent has been activated byor bound to the biological target.

After an agent has been designed, synthesized, and optionallyformulated, it can be tested in vitro by one skilled in the art toassess its biological and performance characteristics. For instance,different types of cells grown in culture can be used to assess thebiological and performance characteristics of the agent. Cellularuptake, binding or cellular localization of the agent can be assessedusing techniques known in the art, including, for example, fluorescentmicroscopy, FACS analysis, immunohistochemistry, immunoprecipitation, insitu hybridization and Forster resonance energy transfer (FRET) orfluorescence resonance energy transfer. By way of example, the agentscan be contacted with a sample for a period of time and then washed toremove any free agents. The sample can then be viewed using anappropriate detection device such as a fluorescent microscope equippedwith appropriate filters matched to the optical properties of afluorescent agent. Fluorescence microscopy of cells in culture orscintillation counting is also a convenient means for determiningwhether uptake and binding has occurred. Tissues, tissue sections andother types of samples such as cytospin samples can also be used in asimilar manner to assess the biological and performance characteristicsof the agents. Other detection methods including, but not limited toflow cytometry, immunoassays, hybridization assays, and microarrayanalysis can also be used.

The invention will now be illustrated by the following examples, whichare given for the purpose of illustration only and without any intentionof limiting the scope of the present invention.

EXAMPLES

Representative materials and methods that may be used in preparing thematerials of the invention are described below. All chemicals andsolvents (reagent grade) were used as commercially obtained withoutfurther purification. HPLC Analysis: Analytical RP HPLC was performed ona Waters 2695 system using either a Phenomenex Phenyl-hexyl column (3μ,100×4.6 mm) or C18 column (5μ, 50×4.6 mm) with a flow rate of 1 to 2mL/min. Chromatograms were monitored at 254 nm and 675 nm. PreparativeHPLC was performed on a Varian system using either a PhenomenexPhenyl-hexyl column or a Phenomenex C18 column (250×10 mm) at 4.7 mL/minusing similar gradient as the analytical run.

Example 1 Synthesis of the Succinimidyl Ester Form of FluorophoreConjugated Peptides

The imaging agents set forth in Table 7 were made in accordance with theprinciples discussed in this Example.

TABLE 7 SEQ ID Imaging Agent NO.Acetyl-Phe-Arg-Lys(F5)-Gly-Gly-Arg-Lys(F5)-OH 385Acetyl-Phe-Gly-Lys(F5)-Arg-Arg-Lys(F5)-Gly-OH 386F5-Gly-Phe-Leu-Gly-Lys(F5)-OH 387Acetyl-Phe-Arg-Lys(F5)-Gly-G-Arg-Lys(F5)-[OH] 388pentynoyl-Phe-Arg-Lys-(F5)-Gly-Gly-Arg-Lys(F5)- 389 [OH]pentynoyl-Phe-Gly-Lys(F5)-Arg-Arg-Lys(F5)-[OH] 390pentynoyl-Lys(F5)-Pro-Leu-Gly-Val-Arg-Lys(F5)- 391 [OH]pentynoyl-Lys(F5)-Gly-Phe-Leu-Gly-βA-Lys(F5)- 392 [OH]pentynoyl-Lys(F5)-Val-Arg-Leu-Gly-Pro-Lys(F5)- 393 [OH]pentynoyl-Lys(F5)-Gly-His-Pro-Gly-Gly-Pro-Gln- 394 Gly-Lys(F5)-[OH]pentynoyl-Lys(F5)-His-Pro-Gly-Gly-Pro-Gln- 395 Lys(F5)-[OH][F5]-His-Gly-Pro-Arg-Lys(F5)-[OH] 242[F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 396 Lys(F5)-[NH2][F5]-His-Gly-Pro-Asn-Lys(F5)-[OH] 240[F5]-His-Gly-Pro-Asn-Lys(F5)-His-Gly-Pro-Asn- 243 βA-[OH][F5]-Ile-His-Pro-Phe-His-Leu-Leu-Tyr-His- 397 Lys(F5)-[OH][F5]-Ile-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr- 398 Lys(F5)-[OH]Acetyl-Lys(F5)-Lys-Lys-Lys(F5)-Gly-[OH] 399Acetyl-Lys(F5)-Lys-Lys-Lys(F5)-Lys-Lys-Gly-[OH] 400[F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His- 401 Lys(F5)-[OH]succinyl-Lys(F5)-Gly-Phe-Leu-Gly-Lys(F5)-[NH₂] 402succinyl-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂] 403succinyl-Lys(F5)-Arg-Arg-Lys(F5)-[NH₂] 404succinyl-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂] 405succinyl-Lys(F6)-Ala-Arg-Arg-Lys(F6)-[NH₂] 406succinyl-Lys(F5)-Lys-Lys-Lys(F5)-[NH₂] 407succinyl-Lys(F6)-Arg-Arg-Arg-Lys(F6)-[NH₂] 408[F5]-His-Gly-Pro-Ile-Lys-[OH] 409 [F5]-Asn-Gly-Pro-Ile-Lys-[OH] 410[F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[OH] 411[F5]-Gly-Val-Arg-Leu-Gly-Pro-Lys(F5) = [OH] 412[F5]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F5)-[OH] 413[F6]-His-Gly-Pro-Asn-Lys(F6)-[OH] 414wherein F5 was the fluorophore described in the imaging agent of FormulaQ77, and F6 was the fluorophore described in the imaging agent ofFormula Q88.Part A. Preparation of Fluorophore Conjugated Peptide

Individual peptide (as set forth in Table 7; 0.45 μmol) and fluorophoreNo. 3 from Table 2 (1.0 μmol) were combined in 100 μL ofN,N-dimethylformamide (DMF) with 1 μL of N-methylmorpholine (NMM) and0.25 mg of N,N-dimethylaminopyridine (DMAP). The solution was placed ona rotator shielded from light and rotated at room temperature for 16hours. The labeled peptide was precipitated by addition of 1.5 mL ofmethyl-t-butyl ether (MTBE) followed by centrifugation and decanting ofthe supernatant. The solid peptide was dried briefly under vacuum,dissolved in 200 μL of 0.1 M sodium bicarbonate and purified by HPLCusing a 10% to 35% gradient of acetonitrile in 25 mM triethylammoniumacetate, pH 7, on a 10 mm×250 mm 300 Å C18 column. The purified peptidewas characterized by LC-MS.

Part B. Preparation of the Succinimidyl Ester of the Labeled Peptide atthe C-Terminus

The fluorophore labeled peptide (0.25 μmol) was dissolved in 50 μL DMFcontaining 0.5 mg of disuccinimidyl carbonate (DSC) and 0.5 μL NMM. Thesolution was placed on rotator shielded from light and rotated at roomtemperature for 4 h. The peptide succinimidyl ester was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant and dried briefly under vacuum.

Example 2 Synthesis of mPEG-5kDa Conjugated Peptide

Methoxypolyethylene glycol amine (mPEG-NH₂, Laysan Bio, molecular weight5 kDa, 2.5 mg, 0.50 μmol) was dissolved in 100 μL DMF and 1 μL NMM. Thesolution was added to 0.25 μmol of the labeled peptide succinimidylester (from Example 1) and the solution was placed on rotator shieldedfrom light and rotated at room temperature for 16 hours. The pegylatedpeptide was precipitated by addition of 1.5 mL of MTBE, isolated bycentrifugation and decanting of the supernatant. The pegylated peptidewas dried briefly under vacuum, dissolved in 350 μL water and purifiedby HPLC on a 10 mm×250 mm 300 Å C18 column. The pegylated peptide wascharacterized by RP-18 HPLC and SEC HPLC.

Example 3 Synthesis of mPEG-20kDa Conjugated Peptide

Methoxypolyethylene glycol amine (mPEG-NH₂, Laysan Bio, molecular weight20 kDa, 10 mg, 0.50 μmol) was dissolved in 100 μL DMF and 1 μL NMM. Thesolution was added to 0.25 μmol of the labeled peptide succinimidylester (from Example 1) and the solution was placed on rotator shieldedfrom light and rotated at room temperature for 16 hours. The pegylatedpeptide was precipitated by addition of 1.5 mL of MTBE, isolated bycentrifugation and decanting of the supernatant. The pegylated peptidewas dried briefly under vacuum, dissolved in 350 μL water and purifiedby HPLC on a 10 mm×250 mm 300 Å C18 column. The pegylated peptide wascharacterized by RP-18 HPLC and SEC HPLC.

Example 4 Synthesis of mPEG-30kDa Conjugated Peptide

Methoxypolyethylene glycol amine (mPEG-NH₂, Laysan Bio, molecular weight30 kDa, 15 mg, 0.50 μmol) was dissolved in 100 μL DMF and 1 μL NMM. Thesolution was added to 0.25 μmol of the labeled peptide succinimidylester (from Example 1) and the solution was placed on rotator shieldedfrom light and rotated at room temperature for 16 hours. The pegylatedpeptide was precipitated by addition of 1.5 mL of MTBE, isolated bycentrifugation and decanting of the supernatant. The pegylated peptidewas dried briefly under vacuum, dissolved in 350 μL water and purifiedby HPLC on a 10 mm×250 mm 300 Å C18 column. The pegylated peptide wascharacterized by RP-18 HPLC and SEC HPLC.

Example 5 Synthesis of Pegylated Poly-L-Lysine Conjugated Peptide

The peptide succinimidyl ester (0.25 μmol) from Example 1, was dissolvedin 50 μL DMSO and added to a solution containing approximately400,000-500,000 Da pegylated poly-L-lysine (0.17 μmol) in 1.5 mL HEPESbuffer, pH 7. The solution was rotated at room temperature for 4 hours,then 20 μL of acetic anhydride and 1.0 mL of carbonate/bicarbonatebuffer, pH 9 was added and rotation continued for 2 hours. The polymerconjugated peptide was purified from unreacted peptide by gel filtrationusing P-100 gel (BioRad) and elution with 1×PBS. Purity of the productwas assessed by RP-18 HPLC and SEC HPLC.

Example 6 Synthesis of Pegylated Poly-D-lysine Conjugated Peptide

The peptide succinimidyl ester (0.25 μmol) from Example 1, is dissolvedin 50 μL DMSO and added to a solution containing approximately300,000-500,000 Da pegylated poly-L-lysine (0.17 μmol) in 1.5 mL HEPESbuffer, pH 7. The solution is rotated at room temperature for 4 hours,then 20 μL of acetic anhydride and 1.0 mL of carbonate/bicarbonatebuffer, pH 9 added and rotation continued for 2 hours. The polymerconjugated peptide is purified from unreacted peptide by gel filtrationusing P-100 gel (BioRad) and eluted with 1×PBS. Purity of the product isassessed by RP-18 HPLCand SEC HPLC.

Example 7 Synthesis of a Branched PEG Peptide Conjugate

Methoxypolyethylene glycol amine (mPEG-NH₂, Laysan Bio, molecular weight20 kDa, 100 mg, 5.0 μmol) was dissolved in 1000 μL DMF. The solution wasadded to 175 μL of DMF containing N-fmoc glutamic acid (0.9 mg, 2.5μmol) and 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU, 1.9 mg, 5 μmol) followed by addition of 2.1μL (12 μmol) of diisopropylethylamine. The solution was stirred at roomtemperature for 2 hours, and the resulting product was precipitated byaddition of 10 mL of methyl tertiary-butyl ether. The solvent wasdecanted and the branched PEG was purified by HPLC on a 21 mm×250 mm 300Å C18 column.

The fmoc group of the purified product was deprotected with 20% v/vdiethylamine in DMF for 30 minutes, followed by precipitation withmethyl tertiary-butyl ether to yield amine-functionalized branched PEG.The amine-functionalized branched PEG (15 mg, 0.50 μmol) was dissolvedin 100 μL DMF and 1 μL NMM. The solution was added to 0.25 μmol of thelabeled peptide (from Example 1) along with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.25mg, 1.3 μmol) and hydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 h. The pegylated peptide was precipitated by additionof 1.5 mL of MTBE, isolated by centrifugation and decanting of thesupernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC using a 10% to 85%gradient of acetonitrile in 25 mM triethylammonium bicarbonate, pH 8.5,on a 21 mm×250 mm 300 Å C18 column. The branched PEG conjugated peptidewas further purified by strong anion exchange chromatography. Thebranched PEG conjugated peptide was characterized by RP-18 HPLC and SECHPLC.

Example 8 Synthesis of a Polyvinylpyrrolidone Peptide Conjugate

Carboxy functionalized polyvinylpyrrolidone (6 kDa, Polymersource, 20mg, 3.3 μmol) was dissolved in 200 μL DMF with disuccinimidyl carbonate(5 mg, 20 μmol) and DMAP (0.5 mg, 5 μmol) and rotated at roomtemperature for 4 hours. The succinimidyl ester functionalizedpolyvinylpyrrolidone was precipitated with 1.5 mL of ethyl acetate.

The labeled peptide from Example 1A above (0.75 μmol) along with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 2.5mg, 13 μmol) and hydroxybenzotriazole (HOBT, 2.5 mg, 19 μmol) weredissolved in 750 μL of 0.1 M MES buffer, pH 6. To the solution was added200 μL of 1 M ethylene diamine dihydrochloride in 1 M HEPES buffer, pH 7and the solution was placed on rotator shielded from light and rotatedat room temperature for 16 hours. The amine functionalized peptide waspurified by HPLC.

The amine functionalized peptide (0.5 μmol) described in the paragraphabove, was dissolved in 200 μL DMF containing 20 mg of the succinimidylester functionalized polyvinylpyrrolidone described above along withDMAP (0.5 mg, 5 μmol). The solution was rotated at RT for 24 hours. Thepolyvinylpyrrolidone conjugated peptide was precipitated by addition of1.5 mL of MTBE and isolated by centrifugation and decanting of thesupernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The polyvinylpyrrolidoneconjugated peptide was further purified by strong anion exchangechromatography. The polyvinylpyrrolidone conjugated peptide wascharacterized by RP-18 HPLC and SEC HPLC.

Example 9 Synthesis of an Aminodextran Peptide Conjugate

Aminodextran (Invitrogen, molecular weight 10 kDa, 19 mg, 1.9 μmol) wasdissolved in 150 μL 0.1 mM MES pH 6. The solution was added to 0.75 μmolof the labeled peptide (from Example 1) along with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 2 mg,10 μmol) and hydroxybenzotriazole (HOBT, 2 mg, 15 μmol) and the solutionwas placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The aminodextran conjugated peptide waspurified by HPLC. The aminodextran conjugated peptide was furtherpurified by gel filtration chromatography using Biogel P10 (Biorad) andeluting with 1×PBS. The aminodextran conjugated peptide wascharacterized by RP-18 HPLC and SEC HPLC.

Example 10 Synthesis of an Iron Oxide Nanoparticle Peptide Conjugate

A solution of amine-functionalized iron oxide nanoparticles (16 mg totaliron) in 8 mL 0.1 mM MES pH 6 was combined with 0.5 μmol of the labeledpeptide (from Example 1A above) along with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 5 mg,25 μmol) and hydroxybenzotriazole (HOBT, 5 mg, 38 μmol) and the solutionwas placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The iron oxide nanoparticle conjugated peptidewas purified by gel filtration chromatography using Biogel P100 (Biorad)and eluting with 1×PBS. The aminodextran conjugated peptide wascharacterized by SEC HPLC.

Example 11 Synthesis of Exemplary Imaging Agent

This example describes the synthesis of an exemplary imaging agentdenoted by Formula XXII.

The fluorophore labeled, alkyne-modified peptideLys-Pro-Leu-Gly-Val-Arg-Lys (SEQ ID NO:415) (400 μmol) was combined withthe azide 11-azido-3,6,9-trioxaundecan-1-amine (azido-PEG-amine, n=3) in200 μL of 65 mM triethylammonium chloride, 25 mM sodium phosphate, pH 5to which 0.2 mg of nano-sized copper powder was added. The mixture wasrotated at room temperature for 1 hour, filtered, and purified by HPLCon a 10 mm×250 mm 300 Å C18 column. The purified peptide wascharacterized by MALDI MS (calculated 3135.05, found 3135.13).

The resulting amine functionalized peptide (0.16 μmol) was dissolved in200 μL DMSO with 0.35 μmol PolyPEG NHS ester (Warwick Effect Polymers)and 0.2 μL diisopropylethylamine. The solution was rotated at RT for 24hours. The pegylated peptide was precipitated by addition of 1.5 mL ofMTBE, isolated by centrifugation and decanting of the supernatant. Thepegylated peptide was dried briefly under vacuum, dissolved in 350 μLwater and purified by HPLC on a 10 mm×250 mm 300 Å C18 column. Thepegylated peptide was characterized by RP-18 HPLC and SEC HPLC.

Example 12 Cyclic Peptide Imaging Agent

This example describes the synthesis of the cyclic imaging agentrepresented by Formula XXIII.

The peptideArg(Pbf)-Arg(Pbf)-Lys(boc)-Nal-Arg(Pbf)-Arg(Pbf)-Lys(boc)-Gly-OH (SEQ IDNO:416) is prepared using solid phase synthesis as known in the art ofsolid phase peptide synthesis using the Fmoc protection/deprotectionstrategy. The resin used is Fmoc-Gly-HMPB-MBHA resin that iscommercially available. The peptide is cleaved off the resin using 1%TFA in methylene chloride to retain acid labile side chain protectinggroups. Head-tail cyclization is effected by heating a solution of thepeptide in DMF in the presence of Hunig's base and HBTU. Aftercyclization is achieved, the side chain protecting groups are removedunder acidic conditions such as 95% TFA-5% water or other similardeprotection cocktails known in the art. Fluorophore 3 from Table 2 iscoupled to the two lysine side chain amines and the product is isolatedand purified by reversed phase HPLC.

Example 13 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the exemplary imaging agent[F5]Lys(COCF₃)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[OH] (SEQ IDNO:417).

Starting material peptideLys(COCF3)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys-(OH) (SEQ ID NO:418) (6μmol) and fluorophore No. 3 from Table 2 (20 μmol) were combined in 2 mLof N,N-dimethylformamide (DMF) with 6 μL of N-methylmorpholine (NMM).The solution was shielded from light and magnetically stirred at roomtemperature for 16 hOURS. The labeled peptide was precipitated byaddition of 2 mL of methyl-t-butyl ether (MTBE) followed bycentrifugation and decanting of the supernatant. The solid peptide wasdried briefly under vacuum, dissolved in 1.2 mL of 0.1 M sodiumbicarbonate and stirred at room temperature for 4 hrs. The mixture thenwas purified by HPLC on a 10 mm×250 mm phenyl hexyl column. The purifiedpeptide was characterized by LC-MS (ESI+, (M+3)/3: 1,131.02).

Example 14 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the exemplary imaging agent[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[OH] (SEQ ID NO:298)(agent R20).

Product agent from Example 13 (2 μmol) was dissolved in 0.9 mL of waterand 0.3 mL of concentrated ammonium hydroxide. The solution was shieldedfrom light and magnetically stirred at room temperature for 16 hours.The mixture then was purified by HPLC on a 10 mm×250 mm phenyl hexylcolumn. The purified peptide was characterized by LC-MS (ESI+, (M+3)/3:1,099.03)

Example 15 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the imaging agent[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[NHmPEG20k (SEQ IDNO:419) (agent R21).

A mixture of product agent from Example 13 (9 μmol), methoxypolyethyleneglycol amine (mPEG-NH₂, Laysan Bio, molecular weight 20 kDa, 220 mg, 11μmol) was dissolved, EDC (52 μmol), HOBt (74 μmol), NMM (40 μL) in 6 mLof DMF and was shielded from light and magnetically stirred at roomtemperature for 3 days. The pegylated peptide was precipitated byaddition of 20 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dissolved in 3 mL of 0.1 Maqueous sodium bicarbonate and stirred at room temperature for 4 hrs.Concentrated ammonium hydroxide (1.2 mL) was added and the resultingmixture was stirred at room temperature for 16 hours. The mixture waspurified by HPLC. The pegylated peptide was further purified by anionexchange chromatography and characterized by RP-HPLC.

Example 16 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the imaging agent[F5]Ile-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Lys(F5)-[OH] (SEQ ID NO:420).

Starting material peptide, Ile-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Lys-(OH)(SEQ ID NO:421) (1.9 μmol) and fluorophore No. 3 from Table 2 (9 μmol)were combined in 900 μL of N,N-dimethylformamide (DMF) with 5 μL ofN-methylmorpholine (NMM). The solution was placed on rotator shieldedfrom light and rotated at room temperature for 16 hours. The labeledpeptide was precipitated by addition of 5 mL of methyl-t-butyl ether(MTBE) followed by centrifugation and decanting of the supernatant. Thesolid peptide was dried briefly under vacuum, dissolved in 0.9 mL of 0.1M hydroxylamine and stirred at room temperature for 2 hrs. To thissolution was added 0.3 mL of 0.1 M aqueous sodium bicarbonate and themixture was stirred at room temperature for 3 days. The mixture waspurified by HPLC. The purified peptide was characterized by LC-MS (ESI+,(M+3)/3: 1,124.03).

Example 17 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the imaging agent[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[NHmPEG5k] (SEQ IDNO:304) (Agent R27)

A mixture of product from Example 13 (2.5 μmol), methoxypolyethyleneglycol amine (mPEG-NH₂, Laysan Bio, molecular weight 5 kDa, 30 mg, 6μmol), EDC (78 μmol), HOBt (111 μmol), NMM (15 μL) in 2 mL of DMF wasshielded from light and magnetically stirred at room temperature for 16hours. The pegylated peptide was precipitated by addition of 10 mL ofMTBE, isolated by centrifugation and decanting of the supernatant. Thepegylated peptide was dissolved in 0.8 mL of 0.1 M aqueous sodiumbicarbonate and stirred at room temperature for 5 hours. Concentratedammonium hydroxide (0.6 mL) was added and the resulting mixture wasstirred at room temperature for 16 hours. The mixture was purified byHPLC using a 10% to 85% gradient of acetonitrile in 25 mMtriethylammonium bicarbonate, pH 8.5, on a 10 mm×250 mm 300 Å C18column. The pegylated peptide was characterized by RP-HPLC.

Example 18 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the imaging agent[F5]Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[NHdPEG₂₄] (SEQ IDNO:305) (Agent R26)

A mixture of product agent from Example 13 (1.0 μmol),methyl-PEG₂₄-Amine (MA(PEG)24 Thermo Scientific, 5.5 μmol), EDC (15μmol), HOBt (17 μmol), NMM (20 μL) in 1 mL of DMF was shielded fromlight and magnetically stirred at room temperature for 16 hours. Thepegylated peptide was precipitated by addition of 10 mL of MTBE,isolated by centrifugation and decanting of the supernatant. Thepegylated peptide was dissolved in 0.7 mL of 0.1 M aqueous sodiumbicarbonate and stirred at room temperature for 2 hours. The maincomponent of the mixture was isolated by preparative HPLC using a 10% to85% gradient of acetonitrile in 25 mM triethylammonium bicarbonate, pH8.5, on a C18 column. The combined fractions were treated withconcentrated ammonium hydroxide (1.0 mL) and the resulting mixture wasstirred at room temperature for 5 days. The mixture was purified by HPLCusing a 10% to 35% gradient of acetonitrile in 25 mM triethylammoniumbicarbonate, pH 8.5 on C18 column. The pegylated peptide wascharacterized by RP-HPLC.

Example 19 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the imaging agent[F5]Lys(Ac)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[OH] (SEQ ID NO:314)(Agent R23)

To a solution of product agent from Example 14 (1.0 μmol) in DMF (1 mL)was added N-methylmorpholine (30 μL) and a solution of succinimidylacetate (2.8 μmol) in acetic acid (4 μL). The solution was placed onrotator shielded from light and rotated at room temperature for 17hours. The peptide was precipitated by addition of ether followed bycentrifugation and decanting of the supernatant. The crude product wasdissolved in 1 mL of 0.1 M aqueous sodium bicarbonate and stirred atroom temperature overnight. The mixture then was purified by HPLC usinga 20% to 35% gradient of acetonitrile in 25 mM triethylammoniumbicarbonate, pH 8.5 on phenyl hexyl column. The purified peptide wascharacterized by LC-MS (ESI+, (M+3)/3: 1,113.03).

Example 20 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the exemplary imaging agent[F5]Lys(COCF₃)-His-Pro-Phe-His-Cha-Val-Ile-His-Lys(F5)-[OH] (SEQ IDNO:458).

Starting material peptideLys(COCF₃)-His-Pro-Phe-His-Cha-Val-Ile-His-Lys-(OH) (SEQ ID NO: 472)(7.8 μmol) and a fluorophore No. 3 of Table 2 (20 μmol) were combined in2 mL of N,N-dimethylformamide (DMF) with 100 μL of N-methylmorpholine(NMM). The solution was shielded from light and magnetically stirred atroom temperature for 16 hours. The labeled peptide was precipitated byaddition of 20 mL of ether followed by centrifugation and decanting ofthe supernatant. The solid peptide was dried briefly under vacuum,dissolved in 1.2 mL of 0.1 M sodium bicarbonate and stirred at roomtemperature for 6 hours. The mixture then was purified by HPLC using a10% to 40% gradient of acetonitrile in 25 mM triethylammoniumbicarbonate, pH 8.5 on a phenyl hexyl column. The purified peptide wascharacterized by LC-MS (ESI+, (M+3)/3: 1,144.37).

Example 21 Synthesis of Exemplary Imaging Agent

This Example describes the synthesis of the exemplary imaging agent[F5]Lys-His-Pro-Phe-His-Cha-Val-Ile-His-Lys(F5)-[OH] (SEQ ID NO:430)(Agent R24).

Combined pure fractions of product from Example 20 (1 μmol, in 4 mL ofHPLC solvent) were combined with 0.4 mL of concentrated ammoniumhydroxide. The solution was shielded from light and stirred at roomtemperature for 16 hours. The mixture was concentrated in vacuo and thenwas purified by HPLC using a 10% to 40% gradient of acetonitrile in 25mM triethylammonium bicarbonate, pH 8.5 on a phenyl hexyl column. Thepurified peptide was characterized by LC-MS (ESI+, (M+3)/3: 1,112.37).

Example 22 Synthesis of Exemplary Imaging Agents

This Example describes the synthesis of the exemplary imaging agentsdenoted as Q65, Q66, Q91, Q92, Q93, Q94, R22, R51, R52, R53, R55, R56,R57, R58 and R59.

(Agent Q65) Pentynoyl-Lys(F5)-Gly-Phe-Leu-Gly-βAla-Lys(F5)-PEG20kDa (SEQID NO:422)

The peptide [pentynoyl]-Lys-Gly-Phe-Leu-Gly-βAla-Lys-[OH] (SEQ IDNO:459) (Tufts University Core Facility, 0.45 μmol) and fluorophore No.3 from Table 2 (1.0 μmol) were combined in 100 μL ofN,N-dimethylformamide (DMF) with 1 μL of N-methylmorpholine (NMM) and0.25 mg of N,N-dimethylaminopyridine (DMAP). The solution was placed ona rotator shielded from light and rotated at room temperature for 16hours. The labeled peptide was precipitated by addition of 1.5 mL ofmethyl-t-butyl ether (MTBE) followed by centrifugation and decanting ofthe supernatant. The solid peptide was dried briefly under vacuum,dissolved in 200 μL of 0.1 M sodium bicarbonate and purified by HPLC.The purified peptide was characterized by LC-MS.

The labeled peptide (25 μmol) then was combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent Q66) Pentynoyl-Lys(F6)-Gly-Phe-Leu-Gly-βAla-Lys(F6)-PEG20kDa (SEQID NO:423)

The peptide [pentynoyl]-Lys-Gly-Phe-Leu-Gly-βAla-Lys-[OH] (SEQ IDNO:460) (Tufts University Core Facility, 0.45 μmol) and fluorophore No.2 (Table 2, 1.0 μmol) were combined in 100 μL of N,N-dimethylformamide(DMF) with 1 μL of N-methylmorpholine (NMM) and 0.25 mg ofN,N-dimethylaminopyridine (DMAP). The solution was placed on a rotatorshielded from light and rotated at room temperature for 16 hours. Thelabeled peptide was precipitated by addition of 1.5 mL of methyl-t-butylether (MTBE) followed by centrifugation and decanting of thesupernatant. The solid peptide was dried briefly under vacuum, dissolvedin 200 μL of 0.1 M sodium bicarbonate and purified by HPLC. The purifiedpeptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent 91) Pentynoyl-Lys(F5)-Pro-Leu-Gly-Val-Arg-Lys(F5)-PEG20kDa (SEQID NO:424)

The peptide [pentynoyl]-Lys-Pro-Leu-Gly-Val-Arg-Lys (SEQ ID NO:461)(Tufts University Core Facility, 0.45 μmol) and fluorophore No. 3 fromTable 2) (1.0 μmol) were combined in 100 μL of N,N-dimethylformamide(DMF) with 1 μL of N-methylmorpholine (NMM) and 0.25 mg ofN,N-dimethylaminopyridine (DMAP). The solution was placed on a rotatorshielded from light and rotated at room temperature for 16 hours. Thelabeled peptide was precipitated by addition of 1.5 mL of methyl-t-butylether (MTBE) followed by centrifugation and decanting of thesupernatant. The solid peptide was dried briefly under vacuum, dissolvedin 200 μL of 0.1 M sodium bicarbonate and purified by HPLC. The purifiedpeptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent Q92) [F5]-His-Gly-Pro-Arg-Lys(F5)-[PEG20kDa] (SEQ ID NO:425)

The peptide [H]-His-Gly-Pro-Arg-Lys-[OH] (SEQ ID NO:85) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore 3 (Table 2, 1.0μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1 μLof N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC on a 10 mm×250 mm 300 Å C18column. The purified peptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 h. The pegylated peptide was precipitated by additionof 1.5 mL of MTBE, isolated by centrifugation and decanting of thesupernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC on a 21 mm×250 mm 300 ÅC18 column. The mPEG conjugated peptide was characterized by RP-18 HPLCand SEC HPLC.

(Agent Q93) [F5]-His-Gly-Pro-Asn-Lys(F5)-His-Gly-Pro-Asn-βA-[PEG20kDa](SEQ ID NO:426)

The peptide [H]-His-Gly-Pro-Asn-Lys-His-Gly-Pro-Asn-[OH] (SEQ ID NO:462)(Tufts University Core Facility, 0.45 μmol) and fluorophore No. 3 fromTable 2 (1.0 μmol) were combined in 100 μL of N,N-dimethylformamide(DMF) with 1 μL of N-methylmorpholine (NMM) and 0.25 mg ofN,N-dimethylaminopyridine (DMAP). The solution was placed on a rotatorshielded from light and rotated at room temperature for 16 hours. Thelabeled peptide was precipitated by addition of 1.5 mL of methyl-t-butylether (MTBE) followed by centrifugation and decanting of thesupernatant. The solid peptide was dried briefly under vacuum, dissolvedin 200 μL of 0.1 M sodium bicarbonate and purified by HPLC. The purifiedpeptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 h. The pegylated peptide was precipitated by additionof 1.5 mL of MTBE, isolated by centrifugation and decanting of thesupernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent Q94) [F5]-His-Gly-Pro-Asn-Lys(F5)-[PEG20kDa] (SEQ ID NO:427)

The peptide [H]-His-Gly-Pro-Asn-Lys-[OH] (SEQ ID NO:75) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 3 from Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent R22) [F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[YPEG 2×20 kDa] (SEQ IDNO:434)

The peptide [H]-Gly-Pro-Leu-Gly-Val-Arg-Lys-[OH] (SEQ ID NO:463) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 2 of Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined Y-PEG-amine 2×20k(Y-shape PEG Amine, MW 40000, JenKem USA, 40 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The Y-PEG conjugatedpeptide was further purified by strong anion exchange chromatography.The Y-PEG conjugated peptide was characterized by RP-18 HPLC and SECHPLC.

(Agent R51) [mPEG-20kDa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂] (SEQID NO:435)

The peptide [succinyl]-Lys-Gly-Phe-Leu-Gly-Lys-[NH₂] (SEQ ID NO:464)(Tufts University Core Facility, 0.45 μmol) and fluorophore No. 2 ofTable 2 (1.0 μmol) were combined in 100 μL of N,N-dimethylformamide(DMF) with 1 μL of N-methylmorpholine (NMM) and 0.25 mg ofN,N-dimethylaminopyridine (DMAP). The solution was placed on a rotatorshielded from light and rotated at room temperature for 16 hours. Thelabeled peptide was precipitated by addition of 1.5 mL of methyl-t-butylether (MTBE) followed by centrifugation and decanting of thesupernatant. The solid peptide was dried briefly under vacuum, dissolvedin 200 μL of 0.1 M sodium bicarbonate and purified by HPLC. The purifiedpeptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent R52) [dPEG-1kDa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂] (SEQID NO:436)

The peptide [succinyl]-Lys-Gly-Phe-Leu-Gly-Lys-[NH₂] (SEQ ID NO:465)(Tufts University Core Facility, 0.45 μmol) and fluorophore No. 2 ofTable 2 (1.0 μmol) were combined in 100 μL of N,N-dimethylformamide(DMF) with 1 μL of N-methylmorpholine (NMM) and 0.25 mg ofN,N-dimethylaminopyridine (DMAP). The solution was placed on a rotatorshielded from light and rotated at room temperature for 16 hours. Thelabeled peptide was precipitated by addition of 1.5 mL of methyl-t-butylether (MTBE) followed by centrifugation and decanting of thesupernatant. The solid peptide was dried briefly under vacuum, dissolvedin 200 μL of 0.1 M sodium bicarbonate and purified by HPLC. The purifiedpeptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with Methyl-PEG₂₄-Amine(MA(PEG)24 Thermo Scientific, 0.54 mg, 0.50 μmol) and dissolved in 100μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC, 0.25 mg, 1.3 μmol) and hydroxybenzotriazole (HOBT,0.2 mg, 1.5 μmol) were added and the solution was placed on rotatorshielded from light and rotated at room temperature for 16 hours. Thediscrete-pegylated peptide was precipitated by addition of 1.5 mL ofMTBE, isolated by centrifugation and decanting of the supernatant. Thediscrete-pegylated peptide was dried briefly under vacuum, dissolved in350 μL water and purified by HPLC. The dPEG conjugated peptide wascharacterized by RP-18 HPLC and LC-MS

(Agent R53) [dppa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂] (SEQ IDNO:437)

The peptide [succinyl]-Lys-Gly-Phe-Leu-Gly-Lys-[NH₂] (SEQ ID NO:466)(Tufts University Core Facility, 0.45 μmol) and fluorophore 2 (Table 2,1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC on a 10 mm×250 mm 300 Å C18column. The purified peptide was characterized by LC-MS.

The labeled peptide (25 μmol) was then combined with3,3-diphenylpropylamine (dppa, 0.11 mg, 0.50 μmol) and dissolved in 100μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC, 0.25 mg, 1.3 μmol) and hydroxybenzotriazole (HOBT,0.2 mg, 1.5 μmol) were added and the solution was placed on rotatorshielded from light and rotated at room temperature for 16 h. The dppaconjugated peptide was precipitated by addition of 1.5 mL of MTBE,isolated by centrifugation and decanting of the supernatant. The dppaconjugated peptide was dried briefly under vacuum, dissolved in 350 μLwater and purified by HPLC. The dppa conjugated peptide wascharacterized by RP-18 HPLC and LC-MS.

(Agent R55) [mPEG-20kDa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂] (SEQ IDNO:438)

The peptide [succinyl]-Lys-Arg-Arg-Lys-[NH₂] (SEQ ID NO:467) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 2 of Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH₂, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent R56) [dPEG-1kDa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂] (SEQ IDNO:439)

The peptide [succinyl]-Lys-Arg-Arg-Lys-[NH₂] (SEQ ID NO:468) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 2 of Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined with Methyl-PEG₂₄-Amine(MA(PEG)24 Thermo Scientific, 0.54 mg, 0.50 μmol) and dissolved in 100μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC, 0.25 mg, 1.3 μmol) and hydroxybenzotriazole (HOBT,0.2 mg, 1.5 μmol) were added and the solution was placed on rotatorshielded from light and rotated at room temperature for 16 hours. Thediscrete-pegylated peptide was precipitated by addition of 1.5 mL ofMTBE, isolated by centrifugation and decanting of the supernatant. Thediscrete-pegylated peptide was dried briefly under vacuum, dissolved in350 μL water and purified by HPLC. The dPEG conjugated peptide wascharacterized by RP-18 HPLC and LC-MS

(Agent R57) [dppa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂] (SEQ ID NO:440)

The peptide [succinyl]-Lys-Arg-Arg-Lys-[NH₂] (SEQ ID NO:469) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 2 of Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined with3,3-diphenylpropylamine (dppa, 0.11 mg, 0.50 μmol) and dissolved in 100μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC, 0.25 mg, 1.3 μmol) and hydroxybenzotriazole (HOBT,0.2 mg, 1.5 μmol) were added and the solution was placed on rotatorshielded from light and rotated at room temperature for 16 hours. Thedppa conjugated peptide was precipitated by addition of 1.5 mL of MTBE,isolated by centrifugation and decanting of the supernatant. The dppaconjugated peptide was dried briefly under vacuum, dissolved in 350 μLwater and purified by HPLC. The dppa conjugated peptide wascharacterized by RP-18 HPLC and LC-MS.

(Agent R58) [mPEG-20kDa-Suc]-Lys(F6)-Ala-Arg-Arg-Lys(F6)-[NH₂] (SEQ IDNO:441)

The peptide [succinyl]-Lys-Ala-Arg-Arg-Lys-[NH₂] (SEQ ID NO:470) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 2 of Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

(Agent R59) [mPEG-20kDa-Suc]-Lys(F6)-Arg-Arg-Arg-Lys(F6)-[NH₂] (SEQ IDNO:442)

The peptide [succinyl]-Lys-Ala-Arg-Arg-Lys-[NH₂] (SEQ ID NO:471) (TuftsUniversity Core Facility, 0.45 μmol) and fluorophore No. 2 of Table 2(1.0 μmol) were combined in 100 μL of N,N-dimethylformamide (DMF) with 1μL of N-methylmorpholine (NMM) and 0.25 mg of N,N-dimethylaminopyridine(DMAP). The solution was placed on a rotator shielded from light androtated at room temperature for 16 hours. The labeled peptide wasprecipitated by addition of 1.5 mL of methyl-t-butyl ether (MTBE)followed by centrifugation and decanting of the supernatant. The solidpeptide was dried briefly under vacuum, dissolved in 200 μL of 0.1 Msodium bicarbonate and purified by HPLC. The purified peptide wascharacterized by LC-MS.

The labeled peptide (25 μmol) was then combined with methoxypolyethylene glycol amine (mPEG-NH2, Laysan Bio, 10 mg, 0.50 μmol) anddissolved in 100 μL DMF with 1 μL NMM. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.25 mg, 1.3 μmol) andhydroxybenzotriazole (HOBT, 0.2 mg, 1.5 μmol) were added and thesolution was placed on rotator shielded from light and rotated at roomtemperature for 16 hours. The pegylated peptide was precipitated byaddition of 1.5 mL of MTBE, isolated by centrifugation and decanting ofthe supernatant. The pegylated peptide was dried briefly under vacuum,dissolved in 350 μL water and purified by HPLC. The mPEG conjugatedpeptide was characterized by RP-18 HPLC and SEC HPLC.

Example 23 Tumor Imaging

NU/NU mice 6-8 weeks old (Charles River Laboratory, Wilmington, Mass.)were injected SC with 2×106 4T-1 or HT-29 cells bilaterally in eachmammary fat pad. When tumors reached an approximate size of 3×3 mm(around 7 days after cell injection), mice were injected intravenouslywith 2-4 nmoles of each imaging agent (5 mice/probe and 2 mice/no probeas control) in 100 μL volume via tail vein. Imaging was conducted at 24hours using a Fluorescence Molecular Tomography system (VisEn Medical,Woburn Mass.).

Pentynoyl-Lys(F5)-Gly-Phe-Leu-Gly-βAla-Lys(F5)-PEG20kDa (SEQ ID NO:422)(Agent Q65) was used to image tumors in vivo. FIG. 1 shows the planarimage of bilateral 4T1 tumors 6 hours after administration of the agent.FIG. 2 shows a fluorescence molecular tomography bilateral image of 4T1tumors 6 hours after administration of agent Q65.

Pentynoyl-Lys(F6)-Gly-Phe-Leu-Gly-βAla-Lys(F6)-PEG20kDa (SEQ ID NO:423)(Agent Q66) was also used to image tumors in vivo. FIG. 3 shows theimage of the 4T1 tumors at 5, 24 and 52 hours after administration ofagent Q66.

Pentynoyl-Lys(F5)-Pro-Leu-Gly-Val-Arg-Lys(F5)-PEG20kDa (SEQ ID NO:424)(Agent Q91) was also used to image tumors in vivo. FIG. 4 shows planarfluorescence reflectance images of HT-29 xenograft tumors at 2, 6 and 24hour after administration of Agent Q91.

Example 24 Enzyme Activation Profile of Imaging Agent 065

In this Example, the enzyme activation profile of imaging agentpentynoyl-Lys(F5)-Gly-Phe-Leu-Gly-βAla-Lys(F5)-PEG20kDa (SEQ ID NO:422)(Agent Q65) was characterized.

The reactions were performed with appropriate buffer at room temperaturein a 96-well plate with a final assay volume of 250 μL and probe andenzyme concentrations of 0.5 μM and 0.05 μM, respectively. The bufferfor Cathepsin B was 25 mM MES (pH 5.0), 1 mM DTT. Recombinant humanCathepsin B (R&D Systems) was activated in buffer of 25 mM MES (pH 5.0)and 5 mM DTT for 15 minutes at room temperature. Recombinant humanCathepsin L (R&D Systems) was activated in buffer of 50 mM MES (pH 6.0),5 mM DTT, for 15 minutes on ice. Recombinant mouse Cathepsin Z (R&DSystems) was activated in buffer of 25 mM NaOAc (pH 3.0), 5 mM DTT for 5minutes at room temperature. The buffer for plasmin (CalBiochem) andtrypsin (Sigma) was PBS at pH 7.4.

The plate was incubated at room temperature and the activity monitoredat 0, 1, 3, 5, and 24 hours with a Gemini (Molecular Devices) platereader at excitation wavelength 663 nm, emission 690 nm, and cut off 665nm. The activity was calculated as the fold of activation by comparingthe fluorescence of the enzyme with probe to the fluorescence of probealone.

The results are shown in FIG. 5, which show that the imaging agent Q65is activated by Cathepsin B but not by plasmin, trypsin and Cathepsin Z.

Example 25 Enzyme Activation Profile of Imaging Agents 092 and 093

In this Example, the enzyme activation profile of imaging agents[F5]-His-Gly-Pro-Arg-Lys(F5)-[PEG20kDa] (SEQ ID NO:425) (Agent Q92) and[F5]-His-Gly-Pro-Asn-Lys(F5)-His-Gly-Pro-Asn-βA-[PEG20kDa] (SEQ IDNO:426) (Agent Q93) were characterized.

The reactions were performed with appropriate buffer at room temperaturein a 96-well plate with a final assay volume of 250 μL and probe andenzyme concentrations of 0.5 μM and 0.05 μM respectively. The buffer foractivated humanized rabbit Cathepsin K (Merck) was 50 mM MES (pH 5.0),2.5 mM EDTA, 2.5 mM DTT. The buffer for Cathepsin B was 25 mM MES (pH5.0), 1 mM DTT. The recombinant human Cathepsin B (R&D Systems) wasactivated in buffer of 25 mM MES (pH 5.0) and 5 mM DTT for 15 minutes atroom temperature. The recombinant human Cathepsin L (R&D Systems) wasactivated in buffer of 50 mM MES (pH 6.0), 5 mM DTT, for 15 minutes onice. The buffer for MMP-2 (BIOMOL) and MMP-9 (BIOMOL) was 50 mM Tris (pH7.5), 10 mM CaCl₂, 150 mM NaCl, and 0.05% Brij-35 detergent. The platewas incubated at room temperature and the activity was monitored at 0,1, 3, 5, and 24 hours with a Gemini (Molecular Devices) plate reader atexcitation wavelength 663 nm, emission 690 nm, and cut off 665 nm. Thereleased fluorescent unit was determined by subtracting the fluorescenceof the probe alone from the total fluorescence.

The results for Q92 are presented in FIG. 6A, and the results for Q93are shown in FIG. 6B, which show that both Q92 and Q93 are activatedprimarily by Cathepsin K.

Example 26 Bone Imaging

BALB/c female mice 7 weeks old (Charles River Laboratory, Wilmington,Mass.) were subjected to ovariectomy or sham surgery to induceosteoporosis. After one to two weeks after surgery, the mice wereinjected intravenously with imaging agent[F5]-His-Gly-Pro-Asn-Lys(F5)-[PEG20kDa] (SEQ ID NO:427) (Agent Q94).Imaging was conducted at various time points using a FluorescenceMolecular Tomography system (VisEn Medical, Bedford, Mass.). The resultsare shown in FIG. 7.

FIG. 7A shows images of an ovariectomized mouse (OVX) compared to acontrol (sham) mouse at 4 and 24 hours post-injection. The quantitationof absolute fluorescence of osteoporosis for the sham and ovariectomizedmice is shown in FIG. 7B. The increased probe signal detected in thetibia region appears to be due to increased Cathepsin K levels in boneloss induced by the ovariectomy procedure.

Example 27 Cardiovascular Disease Imaging

This Example show the imaging of cardiovascular disease using theimaging agent Pentynoyl-Lys(F5)-Gly-Phe-Leu-Gly-βAla-Lys(F5)-PEG20kDa)(SEQ ID NO:422) (Agent Q65).

High cholesterol fed apoE−/− mice were injected with 2 nmol of imagingagent and in vivo imaging was performed with ex vivo fluorescencereflectance imaging. FIG. 8 shows fluorescence reflectance imaging ofcardiovascular disease in inflamed atherosclerotic plaques of mice 24hours post-injection of agent Q65. Fluorescence was detected inplaque-laden vascular sections in the aortic root, the arch and carotidarteries.

Example 28 Inflammation Imaging (Carrageenan Induced Paw Edema)

This Example shows the imaging of inflammation in a mouse model.Briefly, BALB/c female mice 6-8 weeks old (Charles River Laboratory,Wilmington, Mass.) were injected subcutaneously with a 1% solution ofcarrageenan in PBS. After 1 minute post-injection, mice were injectedthe imaging agent Q91. Imaging was conducted at various time pointsusing a Fluorescence Molecular Tomography system (VisEn Medical, WoburnMass.). An image is shown in FIG. 9, which shows that significantly morefluorescence is observed in the mouse paw injected with carrageenancompared to the control paw (no injection).

Example 29 Enzyme Activation Profiles of Agents R20R21, R23, R24, andR27

In this Example, the enzyme activity profiles of the agents([F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[OH]) (SEQ ID NO:401)(R20), ([F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[NHmPEG20k])(SEQ ID NO:428) (R21),([F5]-Lys(Ac)-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[OH]) (SEQ IDNO:429) (R23), ([F5]-Lys-His-Pro-Phe-His-Cha-Val-Ile-His-Lys(F5)-[OH])(SEQ ID NO:430) (R24), and([F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[NHmPEG5k]) (SEQ IDNO:433) (R27) were characterized.

The reactions were performed with appropriate buffer in a 96-well platewith a final assay volume of 250 μL and probe and enzyme concentrationsof 1 μM and 100 nM, respectively. The reactions were incubated at 37 Cfor Renin and room temperature for the other proteases. The buffer forhuman neutrophil Cathepsin G (BIOMOL) was 100 mM Tris (pH 7.5), 1.6 MNaCl. The buffer for Cathepsin D (BIOMOL) was 100 mM Formic Acid (pH3.3). The buffer for recombinant human neutrophil elastase (InnovativeResearch) was 100 mM Tris (pH 7.5). The buffer for plasmin (CalBiochem)and trypsin (Sigma) was 1× phosphate buffered saline (PBS) (pH 7.4). Thebuffer for recombinant human Renin (rhRenin) (Proteos) and recombinantrat Renin (rrRenin) (Proteos) was 50 mM MOPS (pH 7.4), 100 mM NaCl, withfreshly prepared 0.002% tween-20. The plate was incubated at theappropriate temperature and the activity was monitored at 24 hours witha Gemini (Molecular Devices) plate reader at excitation wavelength 663nm, emission 690 nm, and cut off 665 nm. The released fluorescent unitwas determined by subtracting the fluorescence of the probe alone fromthe total fluorescence.

The results are shown in FIG. 11, which show that agents R20, R23, andR24 were activated primarily by recombinant human rennin. Agents R21 andR27 were activated by recombinant human rennin, human neutrophilelastase, human neutrophil cathepsin, and trypsin.

Example 30 Renal Imaging

This example shows the in vivo imaging of kidneys in mice on a controldiet versus a low sodium diet.

C57BL6 mice (Jackson Laboratories) were fed a sodium deficient diet(0.02% Na) and water was replaced with a solution of amiloride (0.1 mg/5mL or 5 mg/kg/day) for 2 days. Control C57BL6 mice were fed regular chowdiet and water. Imaging agents[F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[OH] (SEQ ID NO:401)(R20) and ([F5]-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys(F5)-[NHmPEG20k](SEQ ID NO:428) (R21) were injected intravenously at differentconcentrations (0.2, 0.7, 2 and 4 nmol/mouse) following 2 days on a lowsalt diet. Imaging was conducted at 24 hrs using a FluorescenceMolecular Tomography system (VisEn Medical, Woburn Mass.).

The results are shown in FIG. 12, which demonstrate that for bothimaging agents, the kidneys in the mice on the low sodium diet (FIGS.12B and 12D) gave an increased signal relative to the kidneys in themice on the control diet (FIGS. 12A and 12C).

Example 31 Enzyme Activation of Imaging Agent R22

In this Example, the enzyme activation profile of the imaging agent[F6]-Gly-Pro-Leu-Gly-Val-Arg-Lys(F6)-[YPEG 2×20 kDa] (SEQ ID NO:434)(R22) was characterized.

Activation by recombinant enzymes was carried out in 250 μL volumes setup in 96 well plates. MMPs were activated prior to the reactionaccording to manufacturers' protocols. Reactions contained 0.5 μM of R22and 0.05 μM of activated enzyme in the appropriate activation buffer foreach enzyme and incubated at room temperature for 24 hours. The plateswere read at 24 hours with excitation/emission wavelength 740/770 nm.Fluorescence readings were performed using a Gemini (Molecular Devices)Fluorescence Plate Reader. Wells in which no enzyme was added were usedas controls. Fold activation was obtained by dividing theenzyme-activated read-out (in relative fluorescence units) by itscorresponding control well.

An example of the activation of the R22 by MMPs and other proteases isshown in FIG. 13, which shows that R22 is activated primarily byrecombinant human MMP-13 and trypsin.

Example 32 Tumor Imaging with Agent R22

This Example describes the imaging of tumors in vivo using imaging agentR22.

Male and female NU/NU Mice (Charles River Laboratories, Wilmington,Mass.), 6-8 weeks of age, were injected with tumor cells bilaterally inthe first or second mammary fat pads (human colorectal cancer cell lineHT-29: 3×10⁶ cells/site). Once the tumors reached the desired volume, asmeasured with calipers (100 mm³) mice were injected intravenously with 2nmoles of compound R22. Imaging was performed at 6 and 24 hours laterusing the Fluorescence Tomography System (FMT2500) (VisEn Medical,Bedford, Mass.). For in vivo imaging, mice were anesthetized by gasanesthesia (isoflurane/oxygen mixture), placed in the FMT2500 systemimaging chamber one at a time and imaged using the reflectance andtomographic modes. Examples of imaging with the agent in the HT-29 tumormodel are shown in FIG. 14.

The images taken by reflectance imaging after 6 hours are shown in FIG.14A and after 24 hours in FIG. 14B. Tomographic images taken after 6hours are shown in FIG. 14C and after 24 hours in FIG. 14D.

Example 33 Enzyme Activation Profiles of Agents R51, R52, R55, R56, R57,R58, and R59

This Example shows the enzymatic activation profiles for compounds([mPEG-20kDa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂]) (SEQ ID NO:435)(R51), ([dPEG-1kDa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂]) (SEQ IDNO:436) (R52), ([dppa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH₂]) (SEQID NO:437) (R53), ([mPEG-20kDa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂]) (SEQID NO:438) (R55), ([dPEG-1kDa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂]) (SEQID NO:439) (R56), ([dppa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH₂]) (SEQ IDNO:440) (R57), ([mPEG-20kDa-Suc]-Lys(F6)-Ala-Arg-Arg-Lys(F6)-[NH₂]) (SEQID NO:441) (R58), and([mPEG-20kDa-Suc]-Lys(F6)-Arg-Arg-Arg-Lys(F6)-[NH₂]) (SEQ ID NO:442)(R59).

Compounds at a concentration of 0.5 uM were incubated with Cathepsin B,Cathepsin K, Cathepsin L, or Cathepsin S at a concentration of 0.05 uM.The reaction buffers used were as follows: Cathepsin K—50 mM MES, pH5.0, 2.5 mM EDTA, 2.5 mM DTT; Cathepsin B—25 mM MES, pH 5.0, 1 mM DTT;Cathepsin S—50 mM NaOAc, pH 4.5, 5 mM DTT, 0.25M NaCl; Cathepsin L—50 mMMES, pH 6, 5 mM DTT. The activity was monitored for 24 hours with aGemini (Molecular Devices) fluorescence plate reader at excitation 663nm and emission 690 nm with a cut off of 690 nm. FIG. 15 indicates thatactivation of imaging agent R51 by various Cathepsin proteases isgreater than that of the other imaging agents.

Example 34 Imaging of Macrophage Activity in Atherosclerotic Plaque

Age-matched C57BL6 wild type female mice (Charles river Laboratories)were fed a normal diet and ApoE −/− knockout female mice (JacksonLaboratories) were fed a high cholesterol diet for 10-20 weeks. The micewere injected intravenously with compounds of formula R51([mPEG-20kDa-Suc]-Lys(F6)-Gly-Phe-Leu-Gly-Lys(F6)-[NH2]) (SEQ ID NO:435)and R55 ([mPEG-20kDa-Suc]-Lys(F6)-Arg-Arg-Lys(F6)-[NH2]) (SEQ IDNO:438). The mice were then imaged at 6 and 24 hours by fluorescencemolecular tomography or fluorescence reflectance using an FMT2500 (VisEnMedical, Bedford, Mass.).

FIG. 16 shows an example of fluorescence imaging using compound R51.FIG. 16A shows fluorescence imaging of the aortic arch in ApoE −/− andwild type mice at 6 hours post-injection. FIG. 16B compares thequantified fluorescence of wild type versus ApoE −/− knockout mice at 6hours post-injection with compound R51. FIG. 16C displays thereflectance imaging of the same mice with compound R51 at 6 hours. FIG.16D compares the quantified fluorescence of wild type versus ApoE −/−knockout mice at 6 hours post-injection with compound R51. Imaging agentR51 produced a greater fluorescent signal in the ApoE knockout mice overcontrols demonstrating the use of R51 in imaging cardiovascular diseasemodels.

FIG. 17A shows an example of fluorescence molecular tomographic imagingof mice at 6 hours post-injection with R55. FIG. 17B compares thequantified fluorescence of wild type versus ApoE knockout mice at 6hours post-injection with compound R55. Imaging agent R51 produced agreater fluorescent signal in the ApoE knockout mice over controls,demonstrating the use of R51 in imaging cardiovascular disease models.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications cited herein arehereby expressly incorporated by reference in their entirety and for allpurposes to the same extent as if each was so individually denoted.

EQUIVALENTS

The invention may be embodied in other specific forms without departingform the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. An intramolecularly-quenched imaging agentrepresented by Formula II:M_(m)-[[X]_(r)—X₁*—[X]_(p)—X₂*—[X]_(q)]  (II) wherein: X, independently,for each occurrence, is an amino acid residue; X₁* and X₂* are eachindependently X-L-F; L, independently, for each occurrence, is a linkermoiety or a bond; F is a fluorophore, wherein one fluorophore quenchesthe other fluorophore; M is a biological modifier having a molecularweight between about 5 kDa to about 35 kDa; m is 1 or 2; r is an integerfrom 0 to 4; p is an integer from 1 to 5; q is an integer from 0 to 4;wherein the sum of r, p and q is 2, 3, 4 or 5; the imaging agent havinga molecular weight from about 5 kDa to about 50 kDa and comprising anenzymatic cleavage site located between the fluorophores so that uponenzymatic cleavage, the fluorophores become unquenched and emit afluorescent signal when excited by electromagnetic radiation ofappropriate wavelength.
 2. The agent of claim 1, wherein the amino acidresidue X₂*, X₁* or both X₂* and X₁* is lysine.
 3. The agent of claim 1,wherein F is a far-red or a near-infrared fluorophore.
 4. The agent ofclaim 3, wherein the fluorophore is a carbocyanine fluorophore or anindocyanine fluorophore.
 5. The agent of claim 1, wherein thefluorophore used to create X₁* and X²* is represented by the followinggeneral Formula VII:

or a salt thereof, wherein: X is independently, for each occurrence,selected from the group consisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se; Y₁and Y₂ are independently selected from the group consisting of H and aC₁-C₂₀ aliphatic group optionally substituted with —OR*, N(R*)₂ or —SR*,wherein R* is H or alkyl; W represents a benzo-condensed, anaphtho-condensed or a pyrido-condensed ring; R₁ is selected from thegroup consisting of (CH₂)_(x)CH₃, (CH₂)_(y)SO₃ ⁻ and (CH₂)_(y)SO₃H,wherein x is an integer selected from 0 to 6 and y is an integerselected from 2 to 6; R₂ and R₃ independently are selected, for eachoccurrence, from the group consisting of H, carboxylate, carboxylicacid, carboxylic ester, amine, amide, sulfonamide, hydroxyl, alkoxyl, asulphonic acid moiety and a sulphonate moiety; R₄ is selected from thegroup consisting of (CH₂)_(x)CH₃, (CH₂)_(y)SO₃ ⁻ and (CH₂)_(n)SO₃H,wherein x is an integer selected from 0 to 6 and y is an integerselected from 2 to 6; and Q is selected from a group consisting of aheteroaryl ring substituted with a carboxyl group or 6-memberedheteroaryl ring substituted with a carbonyl group.
 6. The agent of claim1, wherein the fluorophore used to create X₁* and X²* is represented bythe general Formula VIII:

or a salt thereof, wherein: X₁ and X₂ are independently selected, foreach occurrence from the group consisting of C(CH₂K₁)(CH₂K₂), O, S andSe; K₁ and K₂ are independently selected from the group consisting of H,a C₁-C₂₀ aliphatic group optionally substituted with —OR*, N(R*)₂ or—SR*; or K₁ and K₂ together form part of a substituted or unsubstitutedcarbocyclic, or heterocyclic ring; Y₁ and Y₂ are each independently abenzo-condensed ring, a naphtha-condensed ring or a pyrido-condensedring; n₁ is 1, 2, or 3; R₂, R₁₁ and R₁₂ are independently selected fromthe group consisting of H, F, Br, Cl, C₁-C₆ alkyl, C₁-C₆ alkoxy,aryloxy, a nitrogen-containing heterocyclic ring, a nitrogen-containingheteroaromatic ring, a sulfonate, an iminium ion, or any two adjacentR₁₂ and R₁₁ substituents or R₂ and R₁₁ substituents, when taken incombination, form a 4-, 5-, or 6-membered substituted or unsubstitutedcarbocyclic ring, substituted or unsubstituted non-aromatic carbocyclicring or a substituted or unsubstituted carbocyclic aryl ring, whereinthe carbocyclic rings are each independently optionally substituted oneor more times by C₁-C₆ alkyl, halogen, or OR* or SR*; R₁ and R₁₃ are(CH₂)_(x)CH₃, when x is an integer selected from 0 to 6; or R₁ and R₁₃are independently (CH₂)_(n)SO₃ ⁻ or (CH₂)_(n)SO₃H when n is an integerselected from 2 to 6; R₃, R₄ and R₅ are independently selected from thegroup consisting of H, carboxylate, carboxylic acid, carboxylic ester,amine, amide, sulfonamide, hydroxyl, alkoxyl, a sulphonic acid moietyand a sulphonate moiety; Q is absent, or is selected from a carbonylmoiety or a substituted or unsubstituted C₁-C₆ alkyl group, wherein 0-2of the methylene groups of the alkyl group can optionally be replaced byNH, O or S, or a substituted or unsubstituted C₁-C₆ carbocyclic,non-aromatic carbocyclic, heterocyclic or non-aromatic heterocyclic ringwherein the heterocyclic rings contains 1-2 heteroatoms; R₆ is selectedfrom the group consisting of H, a substituted or unsubstituted C₁-C₂₀aliphatic group, a substituted or unsubstituted aryl, a substituted orunsubstituted alkylaryl, wherein R₆ is optionally substituted withhalogen, OR*, N(R*)₂ or SR*, when Q is absent, a carbonyl group, asubstituted or unsubstituted C₁-C₆ alkyl group, wherein 0-2 of themethylene groups of the alkyl group are replaced by NH, O or S, or asubstituted or unsubstituted C₁-C₆ carbocyclic, non-aromaticcarbocyclic, heterocyclic or non-aromatic heterocyclic ring wherein theheterocyclic rings contains 1-2 heteroatoms; or R₆ is H, when Q is acarbonyl; and R₇ is selected from the group consisting of H, asubstituted or unsubstituted C₁-C₂₀ aliphatic group, a substituted orunsubstituted aryl, a substituted or unsubstituted alkylaryl, wherein R₇is optionally substituted with halogen, OR*, N(R*)₂ or SR*; or R₆ andR₇, taken together form a 4-, 5-, 6- or 7-membered heterocyclic ornon-aromatic heterocyclic ring optionally substituted with halogen, OR*,N(R*)₂ or SR*; or NR₆, Q and CHR₇ together form a substituted orunsubstituted or heterocyclic or non-aromatic heterocyclic ring systemwherein the rings contain 1 or 2 heteroatoms, wherein rings areoptionally substituted with —OR*, N(R*)₂ or —SR*; and W is absent or isa group selected from the group consisting of —SO₂NR₆-Q-CHR₇—, —O—,—COO—, and —CONH—; h=0-70; k=0 or 1; d=0-12; m=0-12; p=0-12; Z is a N, Oor S nucleophile functionality moiety or is, or contains a functionalitycapable of reacting with N, O or S nucleophiles; and each R* isindependently H or C₁₋₂₀ alkyl.
 7. The agent of claim 1, wherein Lcomprises a moiety selected from the group consisting of an amido bond,amino-polyethylene glycol-carboxylic acid, amino-polyethylene glycolazide, diaminoPEG, cysteic acid, glutamic acid, aminocaproic acid,ethylenediamine, propylenediamine, spermidine, spermine, hexanediamine,and a diamine-amino acid.
 8. The agent of claim 1, wherein thebiological modifier is selected from the group consisting ofpolyethylene glycol, methoxypolyethylene glycol, branched polypropyleneglycol, polypropylene glycol, a graft copolymer of poly-lysine andmethoxypolyethyleneglycol, a peptide, a carbohydrate, and a dextran. 9.The agent of claim 1, wherein the biological modifier, M is covalentlylinked to the enzymatically cleavable oligopeptide[X]_(r)—X₁*—[X]_(p)—X₂*—[X]_(q) through an acyl moiety.
 10. The agent ofclaim 1, wherein the enzymatic cleavage site is cleavable by at leastone enzyme selected from the group consisting of a cathepsin, a matrixmetalloprotease, an aspartyl protease, a peptidase, a carboxypeptidase,a glycosidase, a lipase, a phospholipase, a phosphatase, aphosphodiesterase, a sulfatase, a reductase, and a bacterial enzyme. 11.The agent of claim 1, wherein each fluorophore F is the same.
 12. Theagent of claim 1, wherein at least one fluorophore when covalentlylinked to the oligopeptide is:


13. The imaging agent of claim 1, wherein[X]_(r)—X₁*—[X]_(p)—X₂*—[X]_(q) is an amino acid sequence selected fromthe group consisting of SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:25; SEQ IDNO:32; SEQ ID NO:50; SEQ ID NO:75; SEQ ID NO:76; SEQ ID NO:85; SEQ IDNO:98; SEQ ID NO:111; SEQ ID NO:150; SEQ ID NO:153; SEQ ID NO:157; SEQID NO:164; SEQ ID NO:176; SEQ ID NO:285; SEQ ID NO:287; SEQ ID NO:411;SEQ ID NO:442; SEQ ID NO:463; SEQ ID NO:464; SEQ ID NO:467; and SEQ IDNO:470.
 14. The imaging agent of claim 1, wherein the imaging agent isselected from the group consisting of: SEQ ID NO:259; SEQ ID NO:260; SEQID NO:273; SEQ ID NO:278, SEQ ID NO:286; SEQ ID NO:289; SEQ ID NO:293;SEQ ID NO:294; SEQ ID NO:424; SEQ ID NO:425; SEQ ID NO:434; SEQ IDNO:435; SEQ ID NO:438; SEQ ID NO:441; and SEQ ID NO:442; wherein F5 isrepresented by the formula

and F6 is represented by the formula


15. The agent of claim 1, wherein F is represented by the formula F6

or a salt thereof.